Methods and compositions for degrading cellulosic material

ABSTRACT

The present invention relates to enzyme compositions comprising a polypeptide having cellobiohydrolase II activity, a polypeptide having xylanase activity, and one or more cellulolytic proteins and their use in the degradation or conversion of cellulosic material.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. application Ser.No. 12/612,401, filed Nov. 4, 2009, now U.S. Pat. No. 8,518,684, whichclaims the benefit of U.S. Provisional Application No. 61/116,605, filedNov. 20, 2008, and U.S. Provisional Application No. 61/174,221, filedApr. 30, 2009, which applications are incorporated herein by reference.

REFERENCE TO A SEQUENCE LISTING

This application contains a Sequence Listing in computer readable form.The computer readable form is incorporated herein by reference.

REFERENCE TO A DEPOSIT OF BIOLOGICAL MATERIAL

This application contains a reference to a deposit of biologicalmaterial, which deposit is incorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to enzyme compositions and methods ofdegrading or converting cellulosic material with the enzymecompositions.

2. Description of the Related Art

Cellulose is a polymer of the simple sugar glucose linked by beta-1,4bonds. Many microorganisms produce enzymes that hydrolyze beta-linkedglucans. These enzymes include endoglucanases, cellobiohydrolases, andbeta-glucosidases. Endoglucanases digest the cellulose polymer at randomlocations, opening it to attack by cellobiohydrolases.Cellobiohydrolases sequentially release molecules of cellobiose from theends of the cellulose polymer. Cellobiose is a water-solublebeta-1,4-linked dimer of glucose. Beta-glucosidases hydrolyze cellobioseto glucose.

The conversion of lignocellulosic feedstocks into ethanol has theadvantages of the ready availability of large amounts of feedstock, thedesirability of avoiding burning or land filling the materials, and thecleanliness of the ethanol fuel. Wood, agricultural residues, herbaceouscrops, and municipal solid wastes have been considered as feedstocks forethanol production. These materials primarily consist of cellulose,hemicellulose, and lignin. Once the cellulose is converted to glucose,the glucose is easily fermented by yeast into ethanol.

There is a need in the art to improve cellulolytic protein compositionsthrough supplementation with additional enzymes to increase efficiencyand to provide cost-effective enzyme solutions for degradation oflignocellulose.

WO 2004/056981 discloses a partial cellobiohydrolase from Myceliophtherathermophila. WO 2008/008070 discloses a cellobiohydrolase fromChrysosporium lucknowense. WO 94/021785 discloses a Family 10 xylanasefrom Aspergillus aculeatus. Ustinov et al., 2008, Enzyme and MicrobialTechnology 43: 56-65, disclose a Family 10 xylanase from Myceliophtherathermophila.

The present invention relates to improved enzyme compositions fordegrading or converting cellulosic material.

SUMMARY OF THE INVENTION

The present invention relates to enzyme compositions comprising apolypeptide having cellobiohydrolase II activity, a polypeptide havingxylanase activity, and one or more (several) cellulolytic proteinsselected from the group consisting of an endoglucanase, acellobiohydrolase, and a beta-glucosidase, wherein one or both of thepolypeptide having cellobiohydrolase II activity and the polypeptidehaving xylanase activity are foreign to the one or more (several)cellulolytic proteins.

The present invention also relates to methods for degrading orconverting a cellulosic material, comprising: treating the cellulosicmaterial with an enzyme composition comprising a polypeptide havingcellobiohydrolase II activity, a polypeptide having xylanase activity,and one or more (several) cellulolytic proteins selected from the groupconsisting of an endoglucanase, a cellobiohydrolase, and abeta-glucosidase, wherein one or both of the polypeptide havingcellobiohydrolase II activity and the polypeptide having xylanaseactivity are foreign to the one or more (several) cellulolytic proteins.

The present invention also relates to methods for producing afermentation product, comprising:

(a) saccharifying a cellulosic material with an enzyme compositioncomprising a polypeptide having cellobiohydrolase II activity, apolypeptide having xylanase activity, and one or more (several)cellulolytic proteins selected from the group consisting of anendoglucanase, a cellobiohydrolase, and a beta-glucosidase, wherein oneor both of the polypeptide having cellobiohydrolase II activity and thepolypeptide having xylanase activity are foreign to the one or more(several) cellulolytic proteins;

(b) fermenting the saccharified cellulosic material with one or more(several) fermenting microorganisms; and

(c) recovering the fermentation product from the fermentation.

The present invention also relates to methods of fermenting a cellulosicmaterial, comprising: fermenting the cellulosic material with one ormore (several) fermenting microorganisms, wherein the cellulosicmaterial is saccharified with an enzyme composition comprising apolypeptide having cellobiohydrolase II activity, a polypeptide havingxylanase activity, and one or more (several) cellulolytic proteinsselected from the group consisting of an endoglucanase, acellobiohydrolase, and a beta-glucosidase, wherein one or both of thepolypeptide having cellobiohydrolase II activity and the polypeptidehaving xylanase activity are foreign to the one or more (several)cellulolytic proteins.

In one aspect, the polypeptide having cellobiohydrolase II activity is aCEL6 polypeptide. In another aspect, the polypeptide having xylanaseactivity is a GH10 polypeptide.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the effect of Myceliophthora thermophila CBS 117.65 Family6 cellobiohydrolases II, Myceliophthora thermophila CBS 202.75 Family 6cellobiohydrolases II, or Aspergillus aculeatus Family 10 xylanase (on a72 hour hydrolysis of PCS (5% w/v) at 50° C. by a fermentation broth ofTrichoderma reesei expressing Thermoascus aurantiacus GH61A polypeptidehaving cellulolytic enhancing activity and an Aspergillus oryzaebeta-glucosidase fusion protein. Each enzyme was added as a 20%replacement (by protein) of the Trichoderma reesei cellulolytic proteincomposition. The dotted line shows the percent conversion by 2 mg of aTrichoderma reesei cellulolytic protein composition per g of cellulose.Enhancement of hydrolysis was demonstrated with mixtures that reachpercent conversion above the dotted line at an equivalent proteinloading. Error bars from triplicate measurements are shown.

FIG. 2 shows the synergistic enhancement of a 72 hour hydrolysis of PCS(5% w/v) at 50° C. by a fermentation broth of Trichoderma reeseiexpressing Thermoascus aurantiacus GH61A polypeptide having cellulolyticenhancing activity and Aspergillus oryzae beta-glucosidase fusionprotein in the presence of combinations of Myceliophthora thermophilaCBS 202.75 CEL6 cellobiohydrolase II (recombinant) or Myceliophthorathermophila CBS 117.65 CEL6 cellobiohydrolase II (native) andAspergillus aculeatus Family 10 xylanase. The mixtures were added as 20%replacements (by protein) of the Trichoderma reesei cellulolytic proteincomposition with a 50:50 mixture of the Myceliophthora thermophila Cel6Acellobiohydrolase II and the Aspergillus aculeatus Family 10 xylanase.The dotted line shows the percent conversion by 2 mg of the Trichodermareesei cellulolytic protein composition per g of cellulose. Enhancementof hydrolysis was demonstrated with mixtures that reach percentconversion above the dotted line at an equivalent protein loading. Errorbars from triplicate measurements are shown.

FIG. 3 shows the effect of Myceliophthora thermophila CBS 117.65 Family6 cellobiohydrolase II, Myceliophthora thermophila CBS 202.75 Family 6cellobiohydrolase II, or Penicillium sp. Family 10 xylanase on a 72 hourhydrolysis of PCS (5% w/v) at 50° C. by a fermentation broth ofTrichoderma reesei expressing Thermoascus aurantiacus GH61A polypeptidehaving cellulolytic enhancing activity and Aspergillus oryzaebeta-glucosidase fusion protein (cellulolytic protein composition). Eachenzyme was added as a 20% replacement (by protein) of the Trichodermareesei cellulolytic protein composition. The dotted line shows thepercent conversion by 2 mg of the Trichoderma reesei cellulolyticprotein composition per g cellulose loading. Enhancement of hydrolysiswas demonstrated with mixtures that reach percent conversion above thedotted line at an equivalent protein loading. Error bars from triplicatemeasurements are shown.

FIG. 4 shows the synergistic enhancement of a 72 hour hydrolysis of PCS(5% w/v) at 50° C. by a fermentation broth of Trichoderma reeseiexpressing Thermoascus aurantiacus GH61A polypeptide having cellulolyticenhancing activity and Aspergillus oryzae beta-glucosidase fusionprotein (cellulolytic protein composition) in the presence ofcombinations of Myceliophthora thermophila CBS 117.65 Family 6cellobiohydrolase II or Myceliophthora thermophila CBS 202.75 Family 6cellobiohydrolase II and Penicillium sp. Family 10 xylanase. Themixtures were added as 20% replacements (by protein) of the Trichodermareesei cellulolytic protein composition with a 50:50 mixture ofMyceliophthora thermophila Cel6A cellobiohydrolase II and Penicilliumsp. xylanase. The dotted line shows the percent conversion by 2 mg ofthe Trichoderma reesei cellulolytic protein composition per g celluloseloading. Enhancement of hydrolysis was demonstrated with mixtures thatreach percent conversion above the dotted line at an equivalent proteinloading. Error bars from triplicate measurements are shown.

FIG. 5 shows the synergistic enhancement of a 72 hour hydrolysis of PCS(5% w/v) at 50° C. by a fermentation broth of Trichoderma reeseiexpressing Thermoascus aurantiacus GH61A polypeptide having cellulolyticenhancing activity and Aspergillus oryzae beta-glucosidase fusionprotein (cellulolytic protein composition) in the presence ofcombinations of Trichoderma reesei Cel6A cellobiohydrolase II orThielavia terrestris Cel6A cellobiohydrolase II and Aspergillusaculeatus Family 10 xylanase. The mixtures were added as 10% additions(by protein) of the Trichoderma reesei cellulolytic enzyme preparationwith Trichoderma reesei Cel6A cellobiohydrolase II, Thielavia terrestrisCel6A cellobiohydrolase II and Aspergillus aculeatus xylanaseseparately, or as mixtures of Trichoderma reesei Cel6A cellobiohydrolaseII or Thielavia terrestris Cel6A cellobiohydrolase II and Aspergillusaculeatus xylanase. The dotted line shows the percent conversion by 2 mgof the Trichoderma reesei cellulolytic protein composition per gcellulose loading. Error bars from triplicate measurements are shown.

DEFINITIONS

Cellobiohydrolase II activity: The term “cellobiohydrolase II activity”is defined herein as a 1,4-D-glucan cellobiohydrolase (E.C. 3.2.1.91)activity that catalyzes the hydrolysis of 1,4-beta-D-glucosidic linkagesin cellulose, cellotetriose, or any beta-1,4-linked glucose containingpolymer, releasing cellobiose from the non-reducing end of the chain.For purposes of the present invention, cellobiohydrolase activity isdetermined according to the procedures described by Lever et al., 1972,Anal. Biochem. 47: 273-279; van Tilbeurgh et al., 1982, FEBS Letters,149: 152-156; van Tilbeurgh and Claeyssens, 1985, FEBS Letters, 187:283-288; and Tomme et al., 1988, Eur. J. Biochem. 170: 575-581. In thepresent invention, the Lever et al. method can be employed to assesshydrolysis of cellulose in corn stover, while the methods of vanTilbeurgh et al. and Tomme et al. can be used to determine thecellobiohydrolase activity on a fluorescent disaccharide derivative.

Cellulolytic activity: The term “cellulolytic activity” is definedherein as a biological activity that hydrolyzes a cellulosic material.The two basic approaches for measuring cellulolytic activity include:(1) measuring the total cellulolytic activity, and (2) measuring theindividual cellulolytic activities (endoglucanases, cellobiohydrolases,and beta-glucosidases) as reviewed in Zhang et al., Outlook forcellulase improvement: Screening and selection strategies, 2006,Biotechnology Advances 24: 452-481. Total cellulolytic activity isusually measured using insoluble substrates, including Whatman No1filter paper, microcrystalline cellulose, bacterial cellulose, algalcellulose, cotton, pretreated lignocellulose, etc. The most common totalcellulolytic activity assay is the filter paper assay using Whatman No 1filter paper as the substrate. The assay was established by theInternational Union of Pure and Applied Chemistry (IUPAC) (Ghose, 1987,Measurement of cellulase activities, Pure Appl. Chem. 59: 257-68).

For purposes of the present invention, cellulolytic activity isdetermined by measuring the increase in hydrolysis of a cellulosicmaterial by cellulolytic enzyme(s) under the following conditions: 1-20mg of cellulolytic protein/g of cellulose in PCS for 3-7 days at 50-65°C. compared to a control hydrolysis without addition of cellulolyticprotein. Typical conditions are 1 ml reactions, washed or unwashed PCS,5% insoluble solids, 50 mM sodium acetate pH 5, 1 mM MnSO₄, 50-65° C.,72 hours, sugar analysis by AMINEX® HPX-87H column (Bio-RadLaboratories, Inc., Hercules, Calif., USA).

Endoglucanase: The term “endoglucanase” is defined herein as anendo-1,4-(1,3;1,4)-beta-D-glucan 4-glucanohydrolase (E.C. 3.2.1.4),which catalyses endohydrolysis of 1,4-beta-D-glycosidic linkages incellulose, cellulose derivatives (such as carboxymethyl cellulose andhydroxyethyl cellulose), lichenin, beta-1,4 bonds in mixed beta-1,3glucans such as cereal beta-D-glucans or xyloglucans, and other plantmaterial containing cellulosic components. Endoglucanase activity can bedetermined based on a reduction in substrate viscosity or increase inreducing ends determined by a reducing sugar assay (Zhang et al., 2006,Biotechnology Advances 24: 452-481). For purposes of the presentinvention, endoglucanase activity is determined using carboxymethylcellulose (CMC) hydrolysis according to the procedure of Ghose, 1987,Pure and Appl. Chem. 59: 257-268.

Beta-glucosidase: The term “beta-glucosidase” is defined herein as abeta-D-glucoside glucohydrolase (E.C. 3.2.1.21), which catalyzes thehydrolysis of terminal non-reducing beta-D-glucose residues with therelease of beta-D-glucose. For purposes of the present invention,beta-glucosidase activity is determined according to the basic proceduredescribed by Venturi et al., 2002, Extracellular beta-D-glucosidase fromChaetomium thermophilum var. coprophilum: production, purification andsome biochemical properties, J. Basic Microbiol. 42: 55-66. One unit ofbeta-glucosidase activity is defined as 1.0 μmole of p-nitrophenolproduced per minute at 40° C., pH 5 from 1 mMp-nitrophenyl-beta-D-glucopyranoside as substrate in 100 mM sodiumcitrate containing 0.01% TWEEN® 20.

Cellulolytic enhancing activity: The term “cellulolytic enhancingactivity” is defined herein as a biological activity that enhances thehydrolysis of a cellulosic material by polypeptides having cellulolyticactivity. For purposes of the present invention, cellulolytic enhancingactivity is determined by measuring the increase in reducing sugars orin the increase of the total of cellobiose and glucose from thehydrolysis of a cellulosic material by cellulolytic protein under thefollowing conditions: 1-50 mg of total protein/g of cellulose in PCS,wherein total protein is comprised of 50-99.5% w/w cellulolytic proteinand 0.5-50% w/w protein of cellulolytic enhancing activity for 1-7 dayat 50-65° C. compared to a control hydrolysis with equal total proteinloading without cellulolytic enhancing activity (1-50 mg of cellulolyticprotein/g of cellulose in PCS). In a preferred aspect, a mixture ofCELLUCLAST® 1.5 L (Novozymes A/S, Bagsværd, Denmark) in the presence of3% of total protein weight Aspergillus oryzae beta-glucosidase(recombinantly produced in Aspergillus oryzae according to WO 02/095014)or 3% of total protein weight Aspergillus fumigatus beta-glucosidase(recombinantly produced in Aspergillus oryzae as described in WO2002/095014) of cellulase protein loading is used as the source of thecellulolytic activity.

The polypeptides having cellulolytic enhancing activity have at least20%, preferably at least 40%, more preferably at least 50%, morepreferably at least 60%, more preferably at least 70%, more preferablyat least 80%, even more preferably at least 90%, most preferably atleast 95%, and even most preferably at least 100% of the cellulolyticenhancing activity of the mature polypeptide of a GH61 polypeptide.

The polypeptides having cellulolytic enhancing activity enhance thehydrolysis of a cellulosic material catalyzed by proteins havingcellulolytic activity by reducing the amount of cellulolytic enzymerequired to reach the same degree of hydrolysis preferably at least1.01-fold, more preferably at least 1.05-fold, more preferably at least1.10-fold, more preferably at least 1.25-fold, more preferably at least1.5-fold, more preferably at least 2-fold, more preferably at least3-fold, more preferably at least 4-fold, more preferably at least5-fold, even more preferably at least 10-fold, and most preferably atleast 20-fold.

Xylan degrading activity: The terms “xylan degrading activity” or“xylanolytic activity” are defined herein as a biological activity thathydrolyzes xylan-containing material. The two basic approaches formeasuring xylanolytic activity include: (1) measuring the totalxylanolytic activity, and (2) measuring the individual xylanolyticactivities (endoxylanases, beta-xylosidases, arabinofuranosidases,alpha-glucuronidases, acetylxylan esterases, feruloyl esterases, andalpha-glucuronyl esterases). Recent progress in assays of xylanolyticenzymes was summarized in several publications including Biely andPuchard, Recent progress in the assays of xylanolytic enzymes, 2006,Journal of the Science of Food and Agriculture 86(11): 1636-1647;Spanikova and Biely, 2006, Glucuronoyl esterase—Novel carbohydrateesterase produced by Schizophyllum commune, FEBS Letters 580(19):4597-4601; Herrmann, Vrsanska, Jurickova, Hirsch, Biely, and Kubicek,1997, The beta-D-xylosidase of Trichoderma reesei is a multifunctionalbeta-D-xylan xylohydrolase, Biochemical Journal 321: 375-381.

Total xylan degrading activity can be measured by determining thereducing sugars formed from various types of xylan, including oat spelt,beechwood, and larchwood xylans, or by photometric determination of dyedxylan fragments released from various covalently dyed xylans. The mostcommon total xylanolytic activity assay is based on production ofreducing sugars from polymeric 4-O-methyl glucuronoxylan as described inBailey, Biely, Poutanen, 1992, Interlaboratory testing of methods forassay of xylanase activity, Journal of Biotechnology 23(3): 257-270.

For purposes of the present invention, xylan degrading activity isdetermined by measuring the increase in hydrolysis of birchwood xylan(Sigma Chemical Co., Inc., St. Louis, Mo., USA) by xylan-degradingenzyme(s) under the following typical conditions: 1 ml reactions, 5mg/ml substrate (total solids), 5 mg of xylanolytic protein/g ofsubstrate, 50 mM sodium acetate pH 5, 50° C., 24 hours, sugar analysisusing p-hydroxybenzoic acid hydrazide (PHBAH) assay as described byLever, 1972, A new reaction for colorimetric determination ofcarbohydrates, Anal. Biochem 47: 273-279.

Xylanase activity: The term “xylanase activity” is defined herein as a1,4-beta-D-xylan-xylohydrolase activity (E.C. 3.2.1.8) that catalyzesthe endo-hydrolysis of 1,4-beta-D-xylosidic linkages in xylans. Forpurposes of the present invention, xylanase activity is determined usingbirchwood xylan as substrate. One unit of xylanase activity is definedas 1.0 μmole of reducing sugar (measured in glucose equivalents asdescribed by Lever, 1972, A new reaction for colorimetric determinationof carbohydrates, Anal. Biochem 47: 273-279) produced per minute duringthe initial period of hydrolysis at 50° C., pH 5 from 2 g of birchwoodxylan per liter as substrate in 50 mM sodium acetate containing 0.01%TWEEN® 20.

Beta-xylosidase activity: The term “beta-xylosidase activity” is definedherein as a beta-D-xyloside xylohydrolase (E.C. 3.2.1.37) that catalyzesthe exo-hydrolysis of short beta (1→4)-xylooligosaccharides, to removesuccessive D-xylose residues from the non-reducing termini. For purposesof the present invention, one unit of beta-xylosidase activity isdefined as 1.0 μmole of p-nitrophenol produced per minute at 40° C., pH5 from 1 mM p-nitrophenyl-beta-D-xyloside as substrate in 100 mM sodiumcitrate containing 0.01% TWEEN® 20.

Acetylxylan esterase activity: The term “acetylxylan esterase activity”is defined herein as a carboxylesterase activity (EC 3.1.1.72) thatcatalyses the hydrolysis of acetyl groups from polymeric xylan,acetylated xylose, acetylated glucose, alpha-napthyl acetate, andp-nitrophenyl acetate. For purposes of the present invention,acetylxylan esterase activity is determined using 0.5 mMp-nitrophenylacetate as substrate in 50 mM sodium acetate pH 5.0containing 0.01% TWEEN™ 20. One unit of acetylxylan esterase activity isdefined as the amount of enzyme capable of releasing 1 μmole ofp-nitrophenolate anion per minute at pH 5, 25° C.

Feruloyl esterase activity: The term “feruloyl esterase activity” isdefined herein as a 4-hydroxy-3-methoxycinnamoyl-sugar hydrolaseactivity (EC 3.1.1.73) that catalyzes the hydrolysis of the4-hydroxy-3-methoxycinnamoyl (feruloyl) group from an esterified sugar,which is usually arabinose in “natural” substrates, to produce ferulate(4-hydroxy-3-methoxycinnamate). Feruloyl esterase is also known asferulic acid esterase, hydroxycinnamoyl esterase, FAE-III, cinnamoylester hydrolase, FAEA, cinnAE, FAE-I, or FAE-II. For purposes of thepresent invention, feruloyl esterase activity is determined using 0.5 mMp-nitrophenylferulate as substrate in 50 mM sodium acetate pH 5.0. Oneunit of feruloyl esterase activity equals the amount of enzyme capableof releasing 1 μmole of p-nitrophenolate anion per minute at pH 5, 25°C.

Alpha-glucuronidase activity: The term “alpha-glucuronidase activity” isdefined herein as an alpha-D-glucosiduronate glucuronohydrolase activity(EC 3.2.1.139) that catalyzes the hydrolysis of an alpha-D-glucuronosideto D-glucuronate and an alcohol. For purposes of the present invention,alpha-glucuronidase activity is determined according to de Vries, 1998,J. Bacteriol. 180: 243-249. One unit of alpha-glucuronidase activityequals the amount of enzyme capable of releasing 1 μmole of glucuronicor 4-O-methylglucuronic acid per minute at pH 5, 40° C.

Alpha-L-arabinofuranosidase activity: The term“alpha-L-arabinofuranosidase activity” is defined herein as analpha-L-arabinofuranoside arabinofuranohydrolase activity (EC 3.2.1.55)that catalyzes the hydrolysis of terminal non-reducingalpha-L-arabinofuranoside residues in alpha-L-arabinosides. The enzymeactivity acts on alpha-L-arabinofuranosides, alpha-L-arabinanscontaining (1,3)- and/or (1,5)-linkages, arabinoxylans, andarabinogalactans. Alpha-L-arabinofuranosidase is also known asarabinosidase, alpha-arabinosidase, alpha-L-arabinosidase,alpha-arabinofuranosidase, polysaccharide alpha-L-arabinofuranosidase,alpha-L-arabinofuranoside hydrolase, L-arabinosidase, oralpha-L-arabinanase. For purposes of the present invention,alpha-L-arabinofuranosidase activity is determined using 5 mg of mediumviscosity wheat arabinoxylan (Megazyme International Ireland, Ltd.,Bray, Co. Wicklow, Ireland) per ml of 100 mM sodium acetate pH 5 in atotal volume of 200 μl for 30 minutes at 40° C. followed by arabinoseanalysis by AMINEX® HPX-87H column chromatography (Bio-Rad Laboratories,Inc., Hercules, Calif., USA).

Family 6 or 10 or 61, or GH6, GH10, or GH61, or CEL6: The terms “Family6”, “Family 10”, “Family 61”, “GH6”, “GH10”, “GH61”, or “CEL6” aredefined herein as a polypeptide falling into the glycoside hydrolaseFamilies 6, 10, and 61 according to Henrissat B., 1991, A classificationof glycosyl hydrolases based on amino-acid sequence similarities,Biochem. J. 280: 309-316, and Henrissat and Bairoch, 1996, Updating thesequence-based classification of glycosyl hydrolases, Biochem. J. 316:695-696. According to such a classification, SEQ ID NOs: 26, 30, 32, 34,and 38 or the mature polypeptides thereof belong to Family 6 and arepredicted to be a cellobiohydrolase II.

Cellulosic material: The cellulosic material can be any materialcontaining cellulose. The predominant polysaccharide in the primary cellwall of biomass is cellulose, the second most abundant is hemicellulose,and the third is pectin. The secondary cell wall, produced after thecell has stopped growing, also contains polysaccharides and isstrengthened by polymeric lignin covalently cross-linked tohemicellulose. Cellulose is a homopolymer of anhydrocellobiose and thusa linear beta-(1-4)-D-glucan, while hemicelluloses include a variety ofcompounds, such as xylans, xyloglucans, arabinoxylans, and mannans incomplex branched structures with a spectrum of substituents. Althoughgenerally polymorphous, cellulose is found in plant tissue primarily asan insoluble crystalline matrix of parallel glucan chains.Hemicelluloses usually hydrogen bond to cellulose, as well as to otherhemicelluloses, which help stabilize the cell wall matrix.

Cellulose is generally found, for example, in the stems, leaves, hulls,husks, and cobs of plants or leaves, branches, and wood of trees. Thecellulosic material can be, but is not limited to, herbaceous material,agricultural residue, forestry residue, municipal solid waste, wastepaper, and pulp and paper mill residue (see, for example, Wiselogel etal., 1995, in Handbook on Bioethanol (Charles E. Wyman, editor), pp.105-118, Taylor & Francis, Washington D.C.; Wyman, 1994, BioresourceTechnology 50: 3-16; Lynd, 1990, Applied Biochemistry and Biotechnology24/25: 695-719; Mosier et al., 1999, Recent Progress in Bioconversion ofLignocellulosics, in Advances in Biochemical Engineering/Biotechnology,T. Scheper, managing editor, Volume 65, pp. 23-40, Springer-Verlag, NewYork). It is understood herein that the cellulose may be in the form oflignocellulose, a plant cell wall material containing lignin, cellulose,and hemicellulose in a mixed matrix. In a preferred aspect, thecellulosic material is lignocellulose.

In one aspect, the cellulosic material is herbaceous material. Inanother aspect, the cellulosic material is agricultural residue. Inanother aspect, the cellulosic material is forestry residue. In anotheraspect, the cellulosic material is municipal solid waste. In anotheraspect, the cellulosic material is waste paper. In another aspect, thecellulosic material is pulp and paper mill residue.

In another aspect, the cellulosic material is corn stover. In anotheraspect, the cellulosic material is corn fiber. In another aspect, thecellulosic material is corn cob. In another aspect, the cellulosicmaterial is orange peel. In another aspect, the cellulosic material isrice straw. In another aspect, the cellulosic material is wheat straw.In another aspect, the cellulosic material is switch grass. In anotheraspect, the cellulosic material is miscanthus. In another aspect, thecellulosic material is bagasse.

In another aspect, the cellulosic material is microcrystallinecellulose. In another aspect, the cellulosic material is bacterialcellulose. In another aspect, the cellulosic material is algalcellulose. In another aspect, the cellulosic material is cotton linter.In another aspect, the cellulosic material is amorphous phosphoric-acidtreated cellulose. In another aspect, the cellulosic material is filterpaper.

The cellulosic material may be used as is or may be subjected topretreatment, using conventional methods known in the art, as describedherein. In a preferred aspect, the cellulosic material is pretreated.

Pretreated corn stover: The term “PCS” or “Pretreated Corn Stover” isdefined herein as a cellulosic material derived from corn stover bytreatment with heat and dilute sulfuric acid.

Xylan-containing material: The term “xylan-containing material” isdefined herein as any material comprising a plant cell wallpolysaccharide containing a backbone of beta-(1-4)-linked xyloseresidues. Xylans of terrestrial plants are heteropolymers possessing abeta-(1-4)-D-xylopyranose backbone, which is branched by shortcarbohydrate chains. They comprise D-glucuronic acid or its 4-O-methylether, L-arabinose, and/or various oligosaccharides, composed ofD-xylose, L-arabinose, D- or L-galactose, and D-glucose. Xylan-typepolysaccharides can be divided into homoxylans and heteroxylans, whichinclude glucuronoxylans, (arabino)glucuronoxylans,(glucurono)arabinoxylans, arabinoxylans, and complex heteroxylans. See,for example, Ebringerova et al., 2005, Adv. Polym. Sci. 186: 1-67.

In the methods of the present invention, any material containing xylanmay be used. In a preferred aspect, the xylan-containing material islignocellulose.

Isolated polypeptide: The term “isolated polypeptide” as used hereinrefers to a polypeptide that is isolated from a source. In a preferredaspect, the polypeptide is at least 1% pure, preferably at least 5%pure, more preferably at least 10% pure, more preferably at least 20%pure, more preferably at least 40% pure, more preferably at least 60%pure, even more preferably at least 80% pure, and most preferably atleast 90% pure, as determined by SDS-PAGE.

Substantially pure polypeptide: The term “substantially purepolypeptide” denotes herein a polypeptide preparation that contains atmost 10%, preferably at most 8%, more preferably at most 6%, morepreferably at most 5%, more preferably at most 4%, more preferably atmost 3%, even more preferably at most 2%, most preferably at most 1%,and even most preferably at most 0.5% by weight of other polypeptidematerial with which it is natively or recombinantly associated. It is,therefore, preferred that the substantially pure polypeptide is at least92% pure, preferably at least 94% pure, more preferably at least 95%pure, more preferably at least 96% pure, more preferably at least 97%pure, more preferably at least 98% pure, even more preferably at least99% pure, most preferably at least 99.5% pure, and even most preferably100% pure by weight of the total polypeptide material present in thepreparation. The polypeptides of the present invention are preferably ina substantially pure form, i.e., that the polypeptide preparation isessentially free of other polypeptide material with which it is nativelyor recombinantly associated. This can be accomplished, for example, bypreparing the polypeptide by well-known recombinant methods or byclassical purification methods.

Mature polypeptide: The term “mature polypeptide” is defined herein as apolypeptide having enzyme activity that is in its final form followingtranslation and any post-translational modifications, such as N-terminalprocessing, C-terminal truncation, glycosylation, phosphorylation, etc.

Mature polypeptide coding sequence: The term “mature polypeptide codingsequence” is defined herein as a nucleotide sequence that encodes amature polypeptide having enzyme activity.

Sequence Identity: The relatedness between two amino acid sequences orbetween two nucleotide sequences is described by the parameter “sequenceidentity”.

For purposes of the present invention, the degree of sequence identitybetween two amino acid sequences is determined using theNeedleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol.48: 443-453) as implemented in the Needle program of the EMBOSS package(EMBOSS: The European Molecular Biology Open Software Suite, Rice etal., 2000, Trends in Genetics 16: 276-277), preferably version 3.0.0 orlater. The optional parameters used are gap open penalty of 10, gapextension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62)substitution matrix. The output of Needle labeled “longest identity”(obtained using the -nobrief option) is used as the percent identity andis calculated as follows:(Identical Residues×100)/(Length of Alignment−Total Number of Gaps inAlignment)

For purposes of the present invention, the degree of sequence identitybetween two deoxyribonucleotide sequences is determined using theNeedleman-Wunsch algorithm (Needleman and Wunsch, 1970, supra) asimplemented in the Needle program of the EMBOSS package (EMBOSS: TheEuropean Molecular Biology Open Software Suite, Rice et al., 2000,supra), preferably version 3.0.0 or later. The optional parameters usedare gap open penalty of 10, gap extension penalty of 0.5, and theEDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix. The outputof Needle labeled “longest identity” (obtained using the -nobriefoption) is used as the percent identity and is calculated as follows:(Identical Deoxyribonucleotides×100)/(Length of Alignment−Total Numberof Gaps in Alignment)

Polypeptide fragment: The term “polypeptide fragment” is defined hereinas a polypeptide having one or more (several) amino acids deleted fromthe amino and/or carboxyl terminus of a mature polypeptide; or ahomologous sequence thereof; wherein the fragment has enzyme activity.

Subsequence: The term “subsequence” is defined herein as a nucleotidesequence having one or more (several) nucleotides deleted from the 5′and/or 3′ end of a mature polypeptide coding sequence; or a homologoussequence thereof; wherein the subsequence encodes a polypeptide fragmenthaving enzyme activity.

Allelic variant: The term “allelic variant” denotes herein any of two ormore alternative forms of a gene occupying the same chromosomal locus.Allelic variation arises naturally through mutation, and may result inpolymorphism within populations. Gene mutations can be silent (no changein the encoded polypeptide) or may encode polypeptides having alteredamino acid sequences. An allelic variant of a polypeptide is apolypeptide encoded by an allelic variant of a gene.

Isolated polynucleotide: The term “isolated polynucleotide” as usedherein refers to a polynucleotide that is isolated from a source. In apreferred aspect, the polynucleotide is at least 1% pure, preferably atleast 5% pure, more preferably at least 10% pure, more preferably atleast 20% pure, more preferably at least 40% pure, more preferably atleast 60% pure, even more preferably at least 80% pure, and mostpreferably at least 90% pure, as determined by agarose electrophoresis.

Substantially pure polynucleotide: The term “substantially purepolynucleotide” as used herein refers to a polynucleotide preparationfree of other extraneous or unwanted nucleotides and in a form suitablefor use within genetically engineered protein production systems. Thus,a substantially pure polynucleotide contains at most 10%, preferably atmost 8%, more preferably at most 6%, more preferably at most 5%, morepreferably at most 4%, more preferably at most 3%, even more preferablyat most 2%, most preferably at most 1%, and even most preferably at most0.5% by weight of other polynucleotide material with which it isnatively or recombinantly associated. A substantially purepolynucleotide may, however, include naturally occurring 5′ and 3′untranslated regions, such as promoters and terminators. It is preferredthat the substantially pure polynucleotide is at least 90% pure,preferably at least 92% pure, more preferably at least 94% pure, morepreferably at least 95% pure, more preferably at least 96% pure, morepreferably at least 97% pure, even more preferably at least 98% pure,most preferably at least 99% pure, and even most preferably at least99.5% pure by weight. The polynucleotides of the present invention arepreferably in a substantially pure form, i.e., that the polynucleotidepreparation is essentially free of other polynucleotide material withwhich it is natively or recombinantly associated. The polynucleotidesmay be of genomic, cDNA, RNA, semisynthetic, synthetic origin, or anycombinations thereof.

Coding sequence: When used herein the term “coding sequence” means anucleotide sequence, which directly specifies the amino acid sequence ofits protein product. The boundaries of the coding sequence are generallydetermined by an open reading frame, which usually begins with the ATGstart codon or alternative start codons such as GTG and TTG and endswith a stop codon such as TAA, TAG, and TGA. The coding sequence may bea DNA, cDNA, synthetic, or recombinant nucleotide sequence.

cDNA: The term “cDNA” is defined herein as a DNA molecule that can beprepared by reverse transcription from a mature, spliced, mRNA moleculeobtained from a eukaryotic cell. cDNA lacks intron sequences that may bepresent in the corresponding genomic DNA. The initial, primary RNAtranscript is a precursor to mRNA that is processed through a series ofsteps before appearing as mature spliced mRNA. These steps include theremoval of intron sequences by a process called splicing. cDNA derivedfrom mRNA lacks, therefore, any intron sequences.

Nucleic acid construct: The term “nucleic acid construct” as used hereinrefers to a nucleic acid molecule, either single- or double-stranded,which is isolated from a naturally occurring gene or which is modifiedto contain segments of nucleic acids in a manner that would nototherwise exist in nature or which is synthetic. The term nucleic acidconstruct is synonymous with the term “expression cassette” when thenucleic acid construct contains the control sequences required forexpression of a coding sequence of the present invention.

Control sequences: The term “control sequences” is defined herein toinclude all components necessary for the expression of a polynucleotideencoding a polypeptide of the present invention. Each control sequencemay be native or foreign to the nucleotide sequence encoding thepolypeptide or native or foreign to each other. Such control sequencesinclude, but are not limited to, a leader, polyadenylation sequence,propeptide sequence, promoter, signal peptide sequence, andtranscription terminator. At a minimum, the control sequences include apromoter, and transcriptional and translational stop signals. Thecontrol sequences may be provided with linkers for the purpose ofintroducing specific restriction sites facilitating ligation of thecontrol sequences with the coding region of the nucleotide sequenceencoding a polypeptide.

Operably linked: The term “operably linked” denotes herein aconfiguration in which a control sequence is placed at an appropriateposition relative to the coding sequence of a polynucleotide sequencesuch that the control sequence directs the expression of the codingsequence of a polypeptide.

Expression: The term “expression” includes any step involved in theproduction of a polypeptide including, but not limited to,transcription, post-transcriptional modification, translation,post-translational modification, and secretion.

Expression vector: The term “expression vector” is defined herein as alinear or circular DNA molecule that comprises a polynucleotide encodinga polypeptide of the present invention and is operably linked toadditional nucleotides that provide for its expression.

Host cell: The term “host cell”, as used herein, includes any cell typethat is susceptible to transformation, transfection, transduction, andthe like with a nucleic acid construct or expression vector comprising apolynucleotide of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to enzyme compositions comprising apolypeptide having cellobiohydrolase II activity, a polypeptide havingxylanase activity, and one or more (several) cellulolytic proteinsselected from the group consisting of an endoglucanase, acellobiohydrolase, and a beta-glucosidase, wherein one or both of thepolypeptide having cellobiohydrolase II activity and the polypeptidehaving xylanase activity are foreign to the one or more (several)cellulolytic proteins.

The present invention also relates to methods for degrading orconverting a cellulosic material, comprising: treating the cellulosicmaterial with an enzyme composition comprising a polypeptide havingcellobiohydrolase II activity, a polypeptide having xylanase activity,and one or more (several) cellulolytic proteins selected from the groupconsisting of an endoglucanase, a cellobiohydrolase, and abeta-glucosidase, wherein one or both of the polypeptide havingcellobiohydrolase II activity and the polypeptide having xylanaseactivity are foreign to the one or more (several) cellulolytic proteins.

In one aspect, the method above further comprises recovering thedegraded or converted cellulosic material. Soluble products ofdegradation or conversion of the cellulosic material can be separatedfrom the insoluble cellulosic material using technology well known inthe art such as, for example, centrifugation, filtration, and gravitysettling.

The present invention also relates to methods for producing afermentation product, comprising: (a) saccharifying a cellulosicmaterial with an enzyme composition comprising a polypeptide havingcellobiohydrolase II activity, a polypeptide having xylanase activity,and one or more (several) cellulolytic proteins selected from the groupconsisting of an endoglucanase, a cellobiohydrolase, and abeta-glucosidase, wherein one or both of the polypeptide havingcellobiohydrolase II activity and the polypeptide having xylanaseactivity are foreign to the one or more (several) cellulolytic proteins;(b) fermenting the saccharified cellulosic material with one or more(several) fermenting microorganisms; and (c) recovering the fermentationproduct from the fermentation.

The present invention also relates to methods of fermenting a cellulosicmaterial, comprising: fermenting the cellulosic material with one ormore (several) fermenting microorganisms, wherein the cellulosicmaterial is saccharified with an enzyme composition comprising apolypeptide having cellobiohydrolase II activity, a polypeptide havingxylanase activity, and one or more (several) cellulolytic proteinsselected from the group consisting of an endoglucanase, acellobiohydrolase, and a beta-glucosidase, wherein one or both of thepolypeptide having cellobiohydrolase II activity and the polypeptidehaving xylanase activity are foreign to the one or more (several)cellulolytic proteins. In a preferred aspect, the fermenting of thecellulosic material produces a fermentation product. In anotherpreferred aspect, the method further comprises recovering thefermentation product from the fermentation.

The presence of the polypeptide having cellobiohydrolase II activity andthe polypeptide having xylanase activity increases the hydrolysis of acellulosic material by the enzyme composition compared to their absenceor the additive effect of each alone. The increase is preferably atleast 1.02-fold, more preferably at least 1.05-fold, more preferably atleast 1.1-fold, more preferably at least 1.2-fold, more preferably atleast 1.4-fold, more preferably at least 1.6-fold, more preferably atleast 1.8-fold, more preferably at least 2-fold, even more preferably atleast 5-fold, and most preferably at least 10-fold in the presence ofthe polypeptide having cellobiohydrolase II activity and the polypeptidehaving xylanase activity compared to their absence or the additiveeffect of each alone.

In one aspect, the polypeptide having cellobiohydrolase II activity isforeign to the one or more (several) cellulolytic proteins. In anotheraspect, the polypeptide having xylanase activity is foreign to the oneor more (several) cellulolytic proteins. In another aspect, thepolypeptide having cellobiohydrolase II activity and the polypeptidehaving xylanase activity are foreign to the one or more (several)cellulolytic proteins.

Enzyme Compositions

In the methods of the present invention, the enzyme compositioncomprises a polypeptide having cellobiohydrolase II activity, apolypeptide having xylanase activity, and one or more (several)cellulolytic proteins selected from the group consisting of anendoglucanase, a cellobiohydrolase, and a beta-glucosidase.

For cellulose degradation, at least three categories of enzymes areimportant for converting cellulose into fermentable sugars:endoglucanases (EC 3.2.1.4) that hydrolyze the cellulose chains atrandom; cellobiohydrolases (EC 3.2.1.91) that cleave cellobiosyl unitsfrom the cellulose chain ends, and beta-glucosidases (EC 3.2.1.21) thatconvert cellobiose and soluble cellodextrins into glucose.

The cellulolytic protein, e.g., endoglucanase, cellobiohydrolase, and/orbeta-glucosidase, may be a bacterial cellulolytic protein. For example,the cellulolytic protein may be a gram positive bacterial cellulolyticprotein such as a Bacillus, Streptococcus, Streptomyces, Staphylococcus,Enterococcus, Lactobacillus, Lactococcus, Clostridium, Geobacillus, orOceanobacillus cellulolytic protein, or a Gram negative bacterialcellulolytic protein such as an E. coli, Pseudomonas, Salmonella,Campylobacter, Helicobacter, Flavobacterium, Fusobacterium, Ilyobacter,Neisseria, or Ureaplasma cellulolytic protein.

In a preferred aspect, the cellulolytic protein is a Bacillusalkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacilluscirculans, Bacillus clausii, Bacillus coagulans, Bacillus firmus,Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillusmegaterium, Bacillus pumilus, Bacillus stearothermophilus, Bacillussubtilis, or Bacillus thuringiensis cellulolytic protein.

In another preferred aspect, the cellulolytic protein is a Streptococcusequisimilis, Streptococcus pyogenes, Streptococcus uberis, orStreptococcus equi subsp. Zooepidemicus cellulolytic protein.

In another preferred aspect, the cellulolytic protein is a Streptomycesachromogenes, Streptomyces avermitilis, Streptomyces coelicolor,Streptomyces griseus, or Streptomyces lividans cellulolytic protein.

The cellulolytic protein, e.g., endoglucanase, cellobiohydrolase, and/orbeta-glucosidase, may also be a fungal cellulolytic protein, and morepreferably a yeast cellulolytic protein such as a Candida,Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowiacellulolytic protein; or more preferably a filamentous fungalcellulolytic protein such as an Acremonium, Agaricus, Alternaria,Aspergillus, Aureobasidium, Botryospaeria, Ceriporiopsis, Chaetomidium,Chrysosporium, Claviceps, Cochliobolus, Coprinopsis, Coptotermes,Corynascus, Cryphonectria, Cryptococcus, Diplodia, Exidia, Filibasidium,Fusarium, Gibberella, Holomastigotoides, Humicola, Irpex, Lentinula,Leptospaeria, Magnaporthe, Melanocarpus, Meripilus, Mucor,Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium,Phanerochaete, Piromyces, Poitrasia, Pseudoplectania,Pseudotrichonympha, Rhizomucor, Schizophyllum, Scytalidium, Talaromyces,Thermoascus, Thielavia, Tolypocladium, Trichoderma, Trichophaea,Verticillium, Volvariella, or Xylaria cellulolytic protein.

In a preferred aspect, the cellulolytic protein is a Saccharomycescarlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus,Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomycesnorbensis, or Saccharomyces oviformis cellulolytic protein.

In another preferred aspect, the cellulolytic protein is an Acremoniumcellulolyticus, Aspergillus aculeatus, Aspergillus awamori, Aspergillusfumigatus, Aspergillus foetidus, Aspergillus japonicus, Aspergillusnidulans, Aspergillus niger, Aspergillus oryzae, Chrysosporiumkeratinophilum, Chrysosporium lucknowense, Chrysosporium tropicum,Chrysosporium merdarium, Chrysosporium inops, Chrysosporium pannicola,Chrysosporium queenslandicum, Chrysosporium zonatum, Fusariumbactridioides, Fusarium cerealis, Fusarium crookwellense, Fusariumculmorum, Fusarium graminearum, Fusarium graminum, Fusariumheterosporum, Fusarium negundi, Fusarium oxysporum, Fusariumreticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum,Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum,Fusarium trichothecioides, Fusarium venenatum, Humicola grisea, Humicolainsolens, Humicola lanuginosa, Irpex lacteus, Mucor miehei,Myceliophthora thermophila, Neurospora crassa, Penicillium funiculosum,Penicillium purpurogenum, Phanerochaete chtysosporium, Thielaviaachromatica, Thielavia albomyces, Thielavia albopilosa, Thielaviaaustraleinsis, Thielavia fimeti, Thielavia microspora, Thielaviaovispora, Thielavia peruviana, Thielavia spededonium, Thielavia setosa,Thielavia subthermophila, Thielavia terrestris, Trichoderma harzianum,Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei,Trichoderma viride, or Trichophaea saccata cellulolytic protein.

The cellulolytic proteins may have activity, i.e., hydrolyze cellulose,either in the acid, neutral, or alkaline pH range. Chemically modifiedor protein engineered mutants of cellulolytic proteins may also be used.

One or more components of the enzyme composition may be wild-typeproteins, recombinant proteins, or a combination of wild-type proteinsand recombinant proteins. For example, one or more components may benative proteins of a cell, which is used as a host cell to expressrecombinantly one or more (several) other components of the enzymecomposition. One or more components of the enzyme composition may beproduced as monocomponents, which are then combined to form the enzymecomposition. The enzyme composition may be a combination ofmulticomponent and monocomponent protein preparations.

Examples of bacterial endoglucanases that can be used in the presentinvention, include, but are not limited to, an Acidothermuscellulolyticus endoglucanase (WO 91/05039; WO 93/15186; U.S. Pat. No.5,275,944; WO 96/02551; U.S. Pat. No. 5,536,655, WO 00/70031, WO05/093050); Thermobifida fusca endoglucanase III (WO 05/093050); andThermobifida fusca endoglucanase V (WO 05/093050).

Examples of fungal endoglucanases that can be used in the presentinvention, include, but are not limited to, a Trichoderma reeseiendoglucanase I (Penttila et al., 1986, Gene 45: 253-263; Trichodermareesei Cel7B endoglucanase I; GENBANK™ accession no. M15665; SEQ ID NO:82); Trichoderma reesei endoglucanase II (Saloheimo, et al., 1988, Gene63:11-22; Trichoderma reesei Cel5A endoglucanase II; GENBANK™ accessionno. M19373; SEQ ID NO: 84); Trichoderma reesei endoglucanase III (Okadaet al., 1988, Appl. Environ. Microbiol. 64: 555-563; GENBANK™ accessionno. AB003694; SEQ ID NO: 86); Trichoderma reesei endoglucanase IV(Saloheimo et al., 1997, Eur. J. Biochem. 249: 584-591; GENBANK™accession no. Y11113; SEQ ID NO: 88); Trichoderma reesei endoglucanase V(Saloheimo et al., 1994, Molecular Microbiology 13: 219-228; GENBANK™accession no. Z33381; SEQ ID NO: 90); Aspergillus aculeatusendoglucanase (Ooi et al., 1990, Nucleic Acids Research 18: 5884);Aspergillus kawachii endoglucanase (Sakamoto et al., 1995, CurrentGenetics 27: 435-439); Erwinia carotovara endoglucanase (Saarilahti etal., 1990, Gene 90: 9-14); Fusarium oxysporum endoglucanase (GENBANK™accession no. L29381); Humicola grisea var. thermoidea endoglucanase(GENBANK™ accession no. AB003107); Melanocarpus albomyces endoglucanase(GENBANK™ accession no. MAL515703); Neurospora crassa endoglucanase(GENBANK™ accession no. XM_(—)324477); Humicola insolens endoglucanase V(SEQ ID NO: 2); Myceliophthora thermophila CBS 117.65 endoglucanase (SEQID NO: 4); basidiomycete CBS 495.95 endoglucanase (SEQ ID NO: 6);basidiomycete CBS 494.95 endoglucanase (SEQ ID NO: 8); Thielaviaterrestris NRRL 8126 CEL6B endoglucanase (SEQ ID NO: 10); Thielaviaterrestris NRRL 8126 CEL6C endoglucanase (SEQ ID NO: 12); Thielaviaterrestris NRRL 8126 CEL7C endoglucanase (SEQ ID NO: 14); Thielaviaterrestris NRRL 8126 CEL7E endoglucanase (SEQ ID NO: 16); Thielaviaterrestris NRRL 8126 CEL7F endoglucanase (SEQ ID NO: 18); Cladorrhinumfoecundissimum ATCC 62373 CEL7A endoglucanase (SEQ ID NO: 20); andTrichoderma reesei strain No. VTT-D-80133 endoglucanase (SEQ ID NO: 22;GENBANK™ accession no. M15665). The endoglucanases of SEQ ID NO: 2, SEQID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22,SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88, and SEQ IDNO: 90 described above are encoded by the mature polypeptide codingsequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17,SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO:85, SEQ ID NO: 87, and SEQ ID NO: 89, respectively.

Examples of cellobiohydrolases useful in the present invention include,but are not limited to, Trichoderma reesei cellobiohydrolase I (SEQ IDNO: 24); Trichoderma reesei cellobiohydrolase II (SEQ ID NO: 26);Humicola insolens cellobiohydrolase I (SEQ ID NO: 28), Myceliophthorathermophila cellobiohydrolase II (SEQ ID NO: 30 and SEQ ID NO: 32),Thielavia terrestris cellobiohydrolase II (CEL6A) (SEQ ID NO: 34),Chaetomium thermophilum cellobiohydrolase I (SEQ ID NO: 36), andChaetomium thermophilum cellobiohydrolase II (SEQ ID NO: 38). Thecellobiohydrolases of SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, and SEQ ID NO:38 described above are encoded by the mature polypeptide coding sequenceof SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ IDNO: 31, SEQ ID NO: 33, SEQ ID NO: 35, and SEQ ID NO: 37, respectively.

Examples of beta-glucosidases useful in the present invention include,but are not limited to, Aspergillus oryzae beta-glucosidase (SEQ ID NO:40); Aspergillus fumigatus beta-glucosidase (SEQ ID NO: 42); Penicilliumbrasilianum IBT 20888 beta-glucosidase (SEQ ID NO: 44); Aspergillusniger beta-glucosidase (SEQ ID NO: 46); and Aspergillus aculeatusbeta-glucosidase (SEQ ID NO: 48). The beta-glucosidases of SEQ ID NO:40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, and SEQ ID NO: 48described above are encoded by the mature polypeptide coding sequence ofSEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, and SEQ IDNO: 47, respectively.

The Aspergillus oryzae polypeptide having beta-glucosidase activity canbe obtained according to WO 2002/095014. The Aspergillus fumigatuspolypeptide having beta-glucosidase activity can be obtained accordingto WO 2005/047499. The Penicillium brasilianum polypeptide havingbeta-glucosidase activity can be obtained according to WO 2007/019442.The Aspergillus niger polypeptide having beta-glucosidase activity canbe obtained according to Dan et al., 2000, J. Biol. Chem. 275:4973-4980. The Aspergillus aculeatus polypeptide having beta-glucosidaseactivity can be obtained according to Kawaguchi et al., 1996, Gene 173:287-288.

Other endoglucanases, cellobiohydrolases, and beta-glucosidases aredisclosed in numerous Glycosyl Hydrolase families using theclassification according to Henrissat B., 1991, A classification ofglycosyl hydrolases based on amino-acid sequence similarities, Biochem.J. 280: 309-316, and Henrissat B., and Bairoch A., 1996, Updating thesequence-based classification of glycosyl hydrolases, Biochem. J. 316:695-696.

In one aspect, the one or more (several) cellulolytic proteins compriseendoglucanase. In another aspect, the one or more (several) cellulolyticproteins comprise endoglucanase I. In another aspect, the one or more(several) cellulolytic proteins comprise endoglucanase II. In anotheraspect, the one or more (several) cellulolytic proteins compriseendoglucanase III. In another aspect, the one or more (several)cellulolytic proteins comprise endoglucanase IV. In another aspect, theone or more (several) cellulolytic proteins comprise endoglucanase V. Inanother aspect, the one or more (several) cellulolytic proteins comprisecellobiohydrolase. In another aspect, the one or more (several)cellulolytic proteins comprise cellobiohydrolase I. In another aspect,the one or more (several) cellulolytic proteins comprisebeta-glucosidase. In another aspect, the one or more (several)cellulolytic proteins comprise a beta-glucosidase fusion protein. Inanother aspect, the one or more (several) cellulolytic proteins compriseendoglucanase and beta-glucosidase. In another aspect, the one or more(several) cellulolytic proteins comprise endoglucanase andcellobiohydrolase I. In another aspect, the one or more (several)cellulolytic proteins comprise endoglucanase, cellobiohydrolase I, andbeta-glucosidase.

In another aspect, the beta-glucosidase is Aspergillus oryzaebeta-glucosidase (SEQ ID NO: 40). In another aspect, thebeta-glucosidase is Aspergillus fumigatus beta-glucosidase (SEQ ID NO:42). In another aspect, the beta-glucosidase is Penicillium brasilianumIBT 20888-beta-glucosidase (SEQ ID NO: 44). In another aspect, thebeta-glucosidase is Aspergillus niger beta-glucosidase (SEQ ID NO: 46).In another aspect, the beta-glucosidase is and Aspergillus aculeatusbeta-glucosidase. In another aspect, the beta-glucosidase is theAspergillus oryzae beta-glucosidase variant fusion protein of SEQ ID NO:50 or the Aspergillus oryzae beta-glucosidase fusion protein of SEQ IDNO: 52. In another aspect, the Aspergillus oryzae beta-glucosidasevariant fusion protein is encoded by the polynucleotide of SEQ ID NO: 49or the Aspergillus oryzae beta-glucosidase fusion protein is encoded bythe polynucleotide of SEQ ID NO: 51.

In another aspect, the one or more (several) cellulolytic proteinscomprise a commercial cellulolytic protein preparation. Commercialcellulolytic protein preparations suitable for use in the presentinvention include, for example, CELLIC™ Ctec (Novozymes NS), CELLUCLAST™(Novozymes A/S), NOVOZYM™ 188 (Novozymes A/S), CELLUZYME™ (NovozymesA/S), CEREFLO™ (Novozymes A/S), and ULTRAFLO™ (Novozymes A/S),ACCELERASE™ (Genencor Int.), LAMINEX™ (Genencor Int.), SPEZYME™ CP(Genencor Int.), ROHAMENT™ 7069 W (Röhm GmbH), FIBREZYME® LDI (DyadicInternational, Inc.), FIBREZYME® LBR (Dyadic International, Inc.), orVISCOSTAR® 150 L (Dyadic International, Inc.). The cellulase enzymes areadded in amounts effective from about 0.001 to about 5.0 wt % of solids,more preferably from about 0.025 to about 4.0 wt % of solids, and mostpreferably from about 0.005 to about 2.0 wt % of solids. The cellulaseenzymes are added in amounts effective from about 0.001 to about 5.0 wt% of solids, more preferably from about 0.025 to about 4.0 wt % ofsolids, and most preferably from about 0.005 to about 2.0 wt % ofsolids.

In another aspect, the one or more (several) cellulolytic proteinscomprise a beta-glucosidase; a Trichoderma reesei cellobiohydrolase I(CEL7A), a Trichoderma reesei cellobiohydrolase II (CEL6A), and aTrichoderma reesei endoglucanase I (CEL7B). In another aspect, the oneor more (several) cellulolytic proteins comprise an Aspergillus oryzaebeta-glucosidase; a Trichoderma reesei cellobiohydrolase I (CEL7A), aTrichoderma reesei cellobiohydrolase II (CEL6A), and a Trichodermareesei endoglucanase I (CEL7B). In another aspect, the one or more(several) cellulolytic proteins comprise an Aspergillus nigerbeta-glucosidase; a Trichoderma reesei cellobiohydrolase I (CEL7A), aTrichoderma reesei cellobiohydrolase II (CEL6A), and a Trichodermareesei endoglucanase I (CEL7B). In another aspect, the one or more(several) cellulolytic proteins comprise an Aspergillus fumigatusbeta-glucosidase; a Trichoderma reesei cellobiohydrolase I (CEL7A), aTrichoderma reesei cellobiohydrolase II (CEL6A), and a Trichodermareesei endoglucanase I (CEL7B). In another aspect, the one or more(several) cellulolytic proteins comprise a Penicillium brasilianumbeta-glucosidase; a Trichoderma reesei cellobiohydrolase I (CEL7A), aTrichoderma reesei cellobiohydrolase II (CEL6A), and a Trichodermareesei endoglucanase I (CEL7B). In another aspect, the one or more(several) cellulolytic proteins comprise an Aspergillus oryzaebeta-glucosidase variant BG fusion protein (for example, SEQ ID NO: 50),a Trichoderma reesei cellobiohydrolase I (CEL7A), a Trichoderma reeseicellobiohydrolase II (CEL6A), and a Trichoderma reesei endoglucanase I(CEL7B). In another aspect, the one or more (several) cellulolyticproteins comprise an Aspergillus oryzae beta-glucosidase fusion protein(for example, SEQ ID NO: 52), a Trichoderma reesei cellobiohydrolase I(CEL7A), a Trichoderma reesei cellobiohydrolase II (CEL6A), and aTrichoderma reesei endoglucanase I (CEL7B).

In another aspect, the one or more (several) cellulolytic proteins abovefurther comprise one or more (several) enzymes selected from the groupconsisting of a Trichoderma reesei endoglucanase II (CEL5A), aTrichoderma reesei endoglucanase V (CEL45A), and a Trichoderma reeseiendoglucanase III (CEL12A).

The enzyme composition may further comprise a polypeptide(s) havingcellulolytic enhancing activity. In another aspect, the one or more(several) cellulolytic proteins above further comprise a polypeptide(s)having cellulolytic enhancing activity comprising the following motifs:

-   -   [I LMV]-P-X(4,5)-G-X-Y-[l LMV]-X-R-X-[EQ]-X(4)-[HNQ] and        [FW]-[TF]-K-[AIV],        wherein X is any amino acid, X(4,5) is any amino acid at 4 or 5        contiguous positions, and X(4) is any amino acid at 4 contiguous        positions.

The isolated polypeptide comprising the above-noted motifs may furthercomprise:

-   -   H-X(1,2)-G-P-X(3)-[YW]-[AILMV],    -   [EQ]-X-Y-X(2)-C-X-[EHQN]-[FILV]-X-[ILV], or    -   H-X(1,2)-G-P-X(3)-[YW]-[AILMV] and        [EQ]-X-Y-X(2)-C-X-[EHQN]-[FILV]-X-[ILV],        wherein X is any amino acid, X(1,2) is any amino acid at 1        position or 2 contiguous positions, X(3) is any amino acid at 3        contiguous positions, and X(2) is any amino acid at 2 contiguous        positions. In the above motifs, the accepted IUPAC single letter        amino acid abbreviation is employed.

In one aspect, the isolated polypeptide having cellulolytic enhancingactivity further comprises H-X(1,2)-G-P-X(3)-[YW]-[AILMV]. In anotheraspect, the isolated polypeptide having cellulolytic enhancing activityfurther comprises [EQ]-X-Y-X(2)-C-X-[EHQN]-[FILV]-X-[ILV]. In anotheraspect, the isolated polypeptide having cellulolytic enhancing activityfurther comprises H-X(1,2)-G-P-X(3)-[YW]-[AILMV] and[EQ]-X-Y-X(2)-C-X-[EHQN]-[FILV]-X-[ILV].

Examples of isolated polypeptides having cellulolytic enhancing activityinclude Thielavia terrestris polypeptides having cellulolytic enhancingactivity (the mature polypeptide of SEQ ID NO: 54, SEQ ID NO: 56, SEQ IDNO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, or SEQ ID NO: 113);Thermoascus aurantiacus (the mature polypeptide of SEQ ID NO: 66);Trichoderma reesei (the mature polypeptide of SEQ ID NO: 68);Myceliophthora thermophila (SEQ ID NO: 115 or SEQ ID NO: 117); orAspergillus fumigatus (SEQ ID NO: 119). The polypeptides havingcellulolytic enhancing activity of SEQ ID NO: 54, SEQ ID NO: 56, SEQ IDNO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQID NO: 68, SEQ ID NO: 113, SEQ ID NO: 113, SEQ ID NO: 115, SEQ ID NO:117, and SEQ ID NO: 119 described above are encoded by the maturepolypeptide coding sequence of SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO:57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ IDNO: 67, SEQ ID NO: 112, SEQ ID NO: 114, SEQ ID NO: 116, and SEQ ID NO:118, respectively.

In one aspect, the one or more (several) cellulolytic proteins furthercomprise a Thielavia terrestris polypeptide having cellulolyticenhancing activity (the mature polypeptide of SEQ ID NO: 54). In anotheraspect, the one or more (several) cellulolytic proteins further comprisea Thielavia terrestris polypeptide having cellulolytic enhancingactivity (the mature polypeptide of SEQ ID NO: 56). In another aspect,the one or more (several) cellulolytic proteins further comprise aThielavia terrestris polypeptide having cellulolytic enhancing activity(the mature polypeptide of SEQ ID NO: 58). In another aspect, the one ormore (several) cellulolytic proteins further comprise a Thielaviaterrestris polypeptide having cellulolytic enhancing activity (themature polypeptide of SEQ ID NO: 60). In another aspect, the one or more(several) cellulolytic proteins further comprise a Thielavia terrestrispolypeptide having cellulolytic enhancing activity (the maturepolypeptide of SEQ ID NO: 62). In another aspect, the one or more(several) cellulolytic proteins further comprise a Thielavia terrestrispolypeptide having cellulolytic enhancing activity (the maturepolypeptide of SEQ ID NO: 64). In another aspect, the one or more(several) cellulolytic proteins further comprise a Thermoascusaurantiacus polypeptide having cellulolytic enhancing activity (themature polypeptide of SEQ ID NO: 66). In another aspect, the one or more(several) cellulolytic proteins further comprise a Trichoderma reeseipolypeptide having cellulolytic enhancing activity (the maturepolypeptide of SEQ ID NO: 68). In another aspect, the one or more(several) cellulolytic proteins further comprise a Thielavia terrestrispolypeptide having cellulolytic enhancing activity (the maturepolypeptide of SEQ ID NO: 113). In another aspect, the one or more(several) cellulolytic proteins further comprise a Myceliophthorathermophila polypeptide having cellulolytic enhancing activity (themature polypeptide of SEQ ID NO: 115). In another aspect, the one ormore (several) cellulolytic proteins further comprise a Myceliophthorathermophila polypeptide having cellulolytic enhancing activity (themature polypeptide of SEQ ID NO: 117). In another aspect, the one ormore (several) cellulolytic proteins further comprise an Aspergillusfumigatus polypeptide having cellulolytic enhancing activity (the maturepolypeptide of SEQ ID NO: 119).

Other cellulolytic enzymes that may be used in the present invention aredescribed in EP 495,257, EP 531,315, EP 531,372, WO 89/09259, WO94/07998, WO 95/24471, WO 96/11262, WO 96/29397, WO 96/034108, WO97/14804, WO 98/08940, WO 98/012307, WO 98/13465, WO 98/015619, WO98/015633, WO 98/028411, WO 99/06574, WO 99/10481, WO 99/025846, WO99/025847, WO 99/031255, WO 2000/009707, WO 2002/050245, WO2002/0076792, WO 2002/101078, WO 2003/027306, WO 2003/052054, WO2003/052055, WO 2003/052056, WO 2003/052057, WO 2003/052118, WO2004/016760, WO 2004/043980, WO 2004/048592, WO 2005/001065, WO2005/028636, WO 2005/093050, WO 2005/093073, WO 2006/074005, WO2006/117432, WO 2007/071818, WO 2007/071820, WO 2008/008070, WO2008/008793, U.S. Pat. No. 4,435,307, U.S. Pat. No. 5,457,046, U.S. Pat.No. 5,648,263, U.S. Pat. No. 5,686,593, U.S. Pat. No. 5,691,178, U.S.Pat. No. 5,763,254, and U.S. Pat. No. 5,776,757.

In another aspect, the enzyme composition may further comprise one ormore xylan-degrading enzymes. In another aspect, the one or morexylan-degrading enzymes are selected from the group consisting of axylanase, an acetyxylan esterase, a feruloyl esterase, anarabinofuranosidase, a xylosidase, and a glucuronidase. In a preferredaspect, the xylosidase is a beta-xylosidase. In a more preferred aspect,the beta-xylosidase is a Trichoderma reesei beta-xylosidase.

Examples of commercial xylan degrading enzyme preparations suitable foruse in the present invention include, for example, SHEARZYME™ (NovozymesA/S), CELLIC™ Htec (Novozymes A/S), VISCOZYME® (Novozymes A/S),ULTRAFLO® (Novozymes A/S), PULPZYME® HC (Novozymes A/S), MULTIFECT®Xylanase (Genencor), ECOPULP® TX-200A (AB Enzymes), HSP 6000 Xylanase(DSM), DEPOL™ 333P (Biocatalysts Limit, Wales, UK), DEPOL™ 740 L.(Biocatalysts Limit, Wales, UK), and DEPOL™ 762P (Biocatalysts Limit,Wales, UK).

Examples of xylanases useful in the methods of the present inventioninclude, but are not limited to, Aspergillus aculeatus xylanase(GeneSeqP:AAR63790; WO 94/21785), Aspergillus fumigatus xylanases (WO2006/078256), and Thielavia terrestris NRRL 8126 xylanases (WO2009/079210).

Examples of beta-xylosidases useful in the methods of the presentinvention include, but are not limited to, Trichoderma reeseibeta-xylosidase (UniProtKB/TrEMBL accession number Q92458), Talaromycesemersonii (SwissProt accession number Q8×212), and Neurospora crassa(SwissProt accession number Q7SOW4).

Examples of acetylxylan esterases useful in the methods of the presentinvention include, but are not limited to, Hypocrea jecorina acetylxylanesterase (WO 2005/001036), Neurospora crassa acetylxylan esterase(UniProt accession number q7s259), Thielavia terrestris NRRL 8126acetylxylan esterase (WO 2009/042846), Chaetomium globosum acetylxylanesterase (Uniprot accession number Q2GWX4), Chaetomium gracileacetylxylan esterase (GeneSeqP accession number AAB82124), Phaeosphaerianodorum acetylxylan esterase (Uniprot accession number Q0UHJ1), andHumicola insolens DSM 1800 acetylxylan esterase (WO 2009/073709).

Examples of ferulic acid esterases useful in the methods of the presentinvention include, but are not limited to, Humicola insolens DSM 1800feruloyl esterase (WO 2009/076122), Neurospora crassa feruloyl esterase(UniProt accession number Q9HGR3), and Neosartorya fischeri feruloylesterase (UniProt Accession number A1D9T4).

Examples of arabinofuranosidases useful in the methods of the presentinvention include, but are not limited to, Humicola insolens DSM 1800arabinofuranosidase (WO 2009/073383) and Aspergillus nigerarabinofuranosidase (GeneSeqP accession number AAR94170).

Examples of alpha-glucuronidases useful in the methods of the presentinvention include, but are not limited to, Aspergillus clavatusalpha-glucuronidase (UniProt accession number alcc12), Trichodermareesei alpha-glucuronidase (Uniprot accession number Q99024),Talaromyces emersonii alpha-glucuronidase (UniProt accession numberQ8×211), Aspergillus niger alpha-glucuronidase (Uniprot accession numberQ96WX9), Aspergillus terreus alpha-glucuronidase (SwissProt accessionnumber Q0CJP9), and Aspergillus fumigatus alpha-glucuronidase (SwissProtaccession number Q4WW45).

An enzyme composition of the present invention may be used as asupplement to another enzyme composition, where the enzyme compositionof the present invention is simply added to the other enzyme compositionor is added as a replacement of a portion of the other enzymecomposition. Replacement of a portion of another enzyme composition,e.g., a commercial preparation, is preferably at least 1%, morepreferably at least 2%, more preferably at least 5%, more preferably atleast 10%, more preferably at least 15%, more preferably at least 20%,more preferably at least 25%, and most preferably at least 50%replacement of the other enzyme composition.

The enzymes and proteins used in the present invention may be fusedpolypeptides or cleavable fusion polypeptides in which anotherpolypeptide is fused at the N-terminus or the C-terminus of thepolypeptide or fragment thereof. A fused polypeptide is produced byfusing a nucleotide sequence (or a portion thereof) encoding anotherpolypeptide to a nucleotide sequence (or a portion thereof) of thepresent invention. Techniques for producing fusion polypeptides areknown in the art, and include ligating the coding sequences encoding thepolypeptides so that they are in frame and that expression of the fusedpolypeptide is under control of the same promoter(s) and terminator.

A fusion polypeptide can further comprise a cleavage site. Uponsecretion of the fusion protein, the site is cleaved releasing thepolypeptide having activity from the fusion protein. Examples ofcleavage sites include, but are not limited to, a Kex2 site that encodesthe dipeptide Lys-Arg (Martin et al., 2003, J. Ind. Microbiol.Biotechnol. 3: 568-576; Svetina et al., 2000, J. Biotechnol. 76:245-251; Rasmussen-Wilson et al., 1997, Appl. Environ. Microbiol. 63:3488-3493; Ward et al., 1995, Biotechnology 13: 498-503; and Contreraset al., 1991, Biotechnology 9: 378-381), an Ile-(Glu or Asp)-Gly-Argsite, which is cleaved by a Factor Xa protease after the arginineresidue (Eaton et al., 1986, Biochem. 25: 505-512); aAsp-Asp-Asp-Asp-Lys site, which is cleaved by an enterokinase after thelysine (Collins-Racie et al., 1995, Biotechnology 13: 982-987); aHis-Tyr-Glu site or His-Tyr-Asp site, which is cleaved by Genenase I(Carter et al., 1989, Proteins: Structure, Function, and Genetics 6:240-248); a Leu-Val-Pro-Arg-Gly-Ser site, which is cleaved by thrombinafter the Arg (Stevens, 2003, Drug Discovery World 4: 35-48); aGlu-Asn-Leu-Tyr-Phe-Gln-Gly site, which is cleaved by TEV protease afterthe Gln (Stevens, 2003, supra); and a Leu-Glu-Val-Leu-Phe-Gln-Gly-Prosite, which is cleaved by a genetically engineered form of humanrhinovirus 3C protease after the Gln (Stevens, 2003, supra).

The enzymes and proteins used in the methods of the present inventionmay be produced by fermentation of the above-noted microbial strains ona nutrient medium containing suitable carbon and nitrogen sources andinorganic salts, using procedures known in the art (see, e.g., Bennett,J. W. and LaSure, L. (eds.), More Gene Manipulations in Fungi, AcademicPress, CA, 1991). Suitable media are available from commercial suppliersor may be prepared according to published compositions (e.g., incatalogues of the American Type Culture Collection). Temperature rangesand other conditions suitable for growth and enzyme production are knownin the art (see, e.g., Bailey, J. E., and 011 is, D. F., BiochemicalEngineering Fundamentals, McGraw-Hill Book Company, NY, 1986).

The fermentation can be any method of cultivation of a cell resulting inthe expression or isolation of an enzyme. Fermentation may, therefore,be understood as comprising shake flask cultivation, or small- orlarge-scale fermentation (including continuous, batch, fed-batch, orsolid state fermentations) in laboratory or industrial fermentorsperformed in a suitable medium and under conditions allowing the enzymeto be expressed or isolated. The resulting enzymes produced by themethods described above may be recovered from the fermentation mediumand purified by conventional procedures.

Polypeptides Having Cellobiohydrolase II Activity and PolynucleotidesThereof

In the methods of the present invention, the polypeptide havingcellobiohydrolase II activity may be obtained from microorganisms of anygenus. In a preferred aspect, the polypeptide obtained from a givensource is secreted extracellularly.

A polypeptide having cellobiohydrolase II activity may be a bacterialpolypeptide. For example, the polypeptide may be a gram positivebacterial polypeptide such as a Bacillus, Streptococcus, Streptomyces,Staphylococcus, Enterococcus, Lactobacillus, Lactococcus, Clostridium,Geobacillus, or Oceanobacillus polypeptide having cellobiohydrolase IIactivity, or a Gram negative bacterial polypeptide such as an E. coli,Pseudomonas, Salmonella, Campylobacter, Helicobacter, Flavobacterium,Fusobacterium, Ilyobacter, Neisseria, or Ureaplasma polypeptide havingcellobiohydrolase II activity.

In a preferred aspect, the polypeptide is a Bacillus alkalophilus,Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans,Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus,Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacilluspumilus, Bacillus stearothermophilus, Bacillus subtilis, or Bacillusthuringiensis polypeptide having cellobiohydrolase II activity.

In another preferred aspect, the polypeptide is a Streptococcusequisimilis, Streptococcus pyogenes, Streptococcus uberis, orStreptococcus equi subsp. Zooepidemicus polypeptide havingcellobiohydrolase II activity.

In another preferred aspect, the polypeptide is a Streptomycesachromogenes, Streptomyces avermitilis, Streptomyces coelicolor,Streptomyces griseus, or Streptomyces lividans polypeptide havingcellobiohydrolase II activity.

The polypeptide having cellobiohydrolase II activity may also be afungal polypeptide, and more preferably a yeast polypeptide such as aCandida, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, orYarrowia polypeptide having cellobiohydrolase II activity; or morepreferably a filamentous fungal polypeptide such as an Acremonium,Agaricus, Alternaria, Aspergillus, Aureobasidium, Botryospaeria,Ceriporiopsis, Chaetomidium, Chrysosporium, Claviceps, Cochliobolus,Coprinopsis, Coptotermes, Corynascus, Cryphonectria, Cryptococcus,Diplodia, Exidia, Filibasidium, Fusarium, Gibberella, Holomastigotoides,Humicola, Irpex, Lentinula, Leptospaeria, Magnaporthe, Melanocarpus,Meripilus, Mucor, Myceliophthora, Neocallimastix, Neurospora,Paecilomyces, Penicillium, Phanerochaete, Piromyces, Poitrasia,Pseudoplectania, Pseudotrichonympha, Rhizomucor, Schizophyllum,Scytalidium, Talaromyces, Thermoascus, Thielavia, Tolypocladium,Trichoderma, Trichophaea, Verticillium, Volvariella, or Xylariapolypeptide having cellobiohydrolase II activity.

In a preferred aspect, the polypeptide is a Saccharomycescarlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus,Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomycesnorbensis, or Saccharomyces oviformis polypeptide havingcellobiohydrolase II activity.

In another preferred aspect, the polypeptide is an Acremoniumcellulolyticus, Aspergillus aculeatus, Aspergillus awamori, Aspergillusfumigatus, Aspergillus foetidus, Aspergillus japonicus, Aspergillusnidulans, Aspergillus niger, Aspergillus oryzae, Chrysosporiumkeratinophilum, Chrysosporium lucknowense, Chrysosporium tropicum,Chrysosporium merdarium, Chrysosporium inops, Chrysosporium pannicola,Chrysosporium queenslandicum, Chrysosporium zonatum, Fusariumbactridioides, Fusarium cerealis, Fusarium crookwellense, Fusariumculmorum, Fusarium graminearum, Fusarium graminum, Fusariumheterosporum, Fusarium negundi, Fusarium oxysporum, Fusariumreticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum,Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum,Fusarium trichothecioides, Fusarium venenatum, Humicola grisea, Humicolainsolens, Humicola lanuginosa, Irpex lacteus, Mucor miehei,Myceliophthora thermophila, Neurospora crassa, Penicillium funiculosum,Penicillium purpurogenum, Phanerochaete chrysosporium, Thielaviaachromatica, Thielavia albomyces, Thielavia albopilosa, Thielaviaaustraleinsis, Thielavia fimeti, Thielavia microspora, Thielaviaovispora, Thielavia peruviana, Thielavia spededonium, Thielavia setosa,Thielavia subthermophila, Thielavia terrestris, Trichoderma harzianum,Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei,Trichoderma viride, or Trichophaea saccata polypeptide havingcellobiohydrolase II activity.

In one aspect, the polypeptide having cellobiohydrolase II activity is aCEL6 polypeptide.

In another aspect, the CEL6 polypeptide having cellobiohydrolase IIactivity is obtained from Myceliophthora thermophila CBS 202.75.

In one aspect, the CEL6 polypeptide having cellobiohydrolase II activitycomprises an amino acid sequence having a degree of identity to themature polypeptide of SEQ ID NO: 30 of preferably at least 60%, morepreferably at least 65%, more preferably at least 70%, more preferablyat least 75%, more preferably at least 80%, more preferably at least85%, even more preferably at least 90%, most preferably at least 95%,and even most preferably at least 96%, at least 97%, at least 98%, or atleast 99%, which have cellobiohydrolase activity (hereinafter“homologous polypeptides”). In another preferred aspect, the homologouspolypeptides comprise amino acid sequences which differ preferably byten amino acids, more preferably by five amino acids, more preferably byfour amino acids, even more preferably by three amino acids, mostpreferably by two amino acids, and even most preferably by one aminoacid from the mature polypeptide of SEQ ID NO: 30.

In another aspect, the CEL6 polypeptide having cellobiohydrolase IIactivity comprises the amino acid sequence of SEQ ID NO: 30 or anallelic variant thereof; or a fragment thereof that hascellobiohydrolase activity. In a preferred aspect, the polypeptidecomprises the amino acid sequence of SEQ ID NO: 30. In another preferredaspect, the polypeptide comprises the mature polypeptide of SEQ ID NO:30. In another preferred aspect, the polypeptide comprises amino acids18 to 482 of SEQ ID NO: 30, or an allelic variant thereof; or a fragmentthereof that has cellobiohydrolase activity. In another preferredaspect, the polypeptide comprises amino acids 18 to 482 of SEQ ID NO:30. In another preferred aspect, the polypeptide consists of the aminoacid sequence of SEQ ID NO: 30 or an allelic variant thereof; or afragment thereof that has cellobiohydrolase activity. In anotherpreferred aspect, the polypeptide consists of the amino acid sequence ofSEQ ID NO: 30. In another preferred aspect, the polypeptide consists ofthe mature polypeptide of SEQ ID NO: 30. In another preferred aspect,the polypeptide consists of amino acids 18 to 482 of SEQ ID NO: 30 or anallelic variant thereof; or a fragment thereof that hascellobiohydrolase activity. In another preferred aspect, the polypeptideconsists of amino acids 18 to 482 of SEQ ID NO: 30.

In another aspect, the CEL6 polypeptide having cellobiohydrolase IIactivity is encoded by a polynucleotide that hybridizes under preferablyvery low stringency conditions, more preferably low stringencyconditions, more preferably medium stringency conditions, morepreferably medium-high stringency conditions, even more preferably highstringency conditions, and most preferably very high stringencyconditions with (i) the mature polypeptide coding sequence of SEQ ID NO:29, (ii) the cDNA sequence contained in the mature polypeptide codingsequence of SEQ ID NO: 29, or (iii) a complementary strand of (i) or(ii). In a preferred aspect, the mature polypeptide coding sequence isnucleotides 52 to 1809 of SEQ ID NO: 29.

In another aspect, the CEL6 polypeptide having cellobiohydrolase IIactivity is encoded by a polynucleotide comprising or consisting of anucleotide sequence that has a degree of identity to the maturepolypeptide coding sequence of SEQ ID NO: 29 of preferably at least 60%,more preferably at least 65%, more preferably at least 70%, morepreferably at least 75%, more preferably at least 80%, more preferablyat least 85%, even more preferably at least 90%, most preferably atleast 95%, and even most preferably at least 96%, at least 97%, at least98%, or at least 99%, which encode a active polypeptide havingcellobiohydrolase II activity.

In another aspect, the CEL6 polypeptide having cellobiohydrolase IIactivity is encoded by a polynucleotide comprising or consisting of thenucleotide sequence of SEQ ID NO: 29. In another more preferred aspect,the nucleotide sequence comprises or consists of the sequence containedin plasmid pSMai182 which is contained in E. coli NRRL B-50059. Inanother preferred aspect, the nucleotide sequence comprises or consistsof the mature polypeptide coding region of SEQ ID NO: 29. In anotherpreferred aspect, the nucleotide sequence comprises or consists ofnucleotides 52 to 1809 of SEQ ID NO: 29. In another more preferredaspect, the nucleotide sequence comprises or consists of the maturepolypeptide coding region contained in plasmid pSMai182 which iscontained in E. coli NRRL B-50059. The present invention alsoencompasses nucleotide sequences which encode a polypeptide comprisingor consisting of the amino acid sequence of SEQ ID NO: 30 or the maturepolypeptide thereof, which differ from SEQ ID NO: 29 or the maturepolypeptide coding sequence thereof by virtue of the degeneracy of thegenetic code. The present invention also relates to subsequences of SEQID NO: 29 which encode fragments of SEQ ID NO: 30 that havecellobiohydrolase activity.

In another preferred aspect, the CEL6 polypeptide havingcellobiohydrolase II activity is obtained from Myceliophthorathermophila CBS 117.65.

In one aspect, the CEL6 polypeptide having cellobiohydrolase II activitycomprises an amino acid sequence having a degree of identity to themature polypeptide of SEQ ID NO: 32 of preferably at least 60%, morepreferably at least 65%, more preferably at least 70%, more preferablyat least 75%, more preferably at least 80%, more preferably at least85%, even more preferably at least 90%, most preferably at least 95%,and even most preferably at least 96%, at least 97%, at least 98%, or atleast 99%, which have cellobiohydrolase activity (hereinafter“homologous polypeptides”). In another preferred aspect, the homologouspolypeptides comprise amino acid sequences which differ preferably byten amino acids, more preferably by five amino acids, more preferably byfour amino acids, even more preferably by three amino acids, mostpreferably by two amino acids, and even most preferably by one aminoacid from the mature polypeptide of SEQ ID NO: 32.

In another aspect, the CEL6 polypeptide having cellobiohydrolase IIactivity comprises the amino acid sequence of SEQ ID NO: 32 or anallelic variant thereof; or a fragment thereof that hascellobiohydrolase activity. In a preferred aspect, the polypeptidecomprises the amino acid sequence of SEQ ID NO: 32. In another preferredaspect, the polypeptide comprises the mature polypeptide of SEQ ID NO:32. In another preferred aspect, the polypeptide comprises amino acids18 to 482 of SEQ ID NO: 32, or an allelic variant thereof; or a fragmentthereof that has cellobiohydrolase activity. In another preferredaspect, the polypeptide comprises amino acids 18 to 482 of SEQ ID NO:32. In another preferred aspect, the polypeptide consists of the aminoacid sequence of SEQ ID NO: 32 or an allelic variant thereof; or afragment thereof that has cellobiohydrolase activity. In anotherpreferred aspect, the polypeptide consists of the amino acid sequence ofSEQ ID NO: 32. In another preferred aspect, the polypeptide consists ofthe mature polypeptide of SEQ ID NO: 32. In another preferred aspect,the polypeptide consists of amino acids 18 to 482 of SEQ ID NO: 32 or anallelic variant thereof; or a fragment thereof that hascellobiohydrolase activity. In another preferred aspect, the polypeptideconsists of amino acids 18 to 482 of SEQ ID NO: 32.

In another aspect, the CEL6 polypeptide having cellobiohydrolase IIactivity is encoded by a polynucleotide that hybridizes under preferablyvery low stringency conditions, more preferably low stringencyconditions, more preferably medium stringency conditions, morepreferably medium-high stringency conditions, even more preferably highstringency conditions, and most preferably very high stringencyconditions with (i) the mature polypeptide coding sequence of SEQ ID NO:31, (ii) the cDNA sequence contained in the mature polypeptide codingsequence of SEQ ID NO: 31, or (iii) a complementary strand of (i) or(ii). In a preferred aspect, the mature polypeptide coding sequence isnucleotides 52 to 1799 of SEQ ID NO: 31.

In another aspect, the CEL6 polypeptide having cellobiohydrolase IIactivity is encoded by a polynucleotide comprising or consisting of anucleotide sequence that has a degree of identity to the maturepolypeptide coding sequence of SEQ ID NO: 31 of preferably at least 60%,more preferably at least 65%, more preferably at least 70%, morepreferably at least 75%, more preferably at least 80%, more preferablyat least 85%, even more preferably at least 90%, most preferably atleast 95%, and even most preferably at least 96%, at least 97%, at least98%, or at least 99%, which encode an active polypeptide.

In another aspect, the CEL6 polypeptide having cellobiohydrolase IIactivity is encoded by a polynucleotide comprising or consisting of thenucleotide sequence of SEQ ID NO: 31. In another preferred aspect, thenucleotide sequence comprises or consists of the mature polypeptidecoding region of SEQ ID NO: 31. In another preferred aspect, thenucleotide sequence comprises or consists of nucleotides 52 to 1799 ofSEQ ID NO: 31. The present invention also encompasses nucleotidesequences which encode a polypeptide comprising or consisting of theamino acid sequence of SEQ ID NO: 32 or the mature polypeptide thereof,which differ from SEQ ID NO: 31 or the mature polypeptide codingsequence thereof by virtue of the degeneracy of the genetic code. Thepresent invention also relates to subsequences of SEQ ID NO: 31 whichencode fragments of SEQ ID NO: 32 that have cellobiohydrolase activity.

In another preferred aspect, the CEL6 polypeptide havingcellobiohydrolase II activity is obtained from Thielavia terrestris NRRL8126.

In one aspect, the CEL6 polypeptide having cellobiohydrolase II activitycomprises an amino acid sequence having a degree of identity to themature polypeptide of SEQ ID NO: 34 of preferably at least 60%, morepreferably at least 65%, more preferably at least 70%, more preferablyat least 75%, more preferably at least 80%, more preferably at least85%, even more preferably at least 90%, most preferably at least 95%,and even most preferably at least 96%, at least 97%, at least 98%, or atleast 99%, which have cellobiohydrolase activity (hereinafter“homologous polypeptides”). In another preferred aspect, the homologouspolypeptides comprise amino acid sequences which differ preferably byten amino acids, more preferably by five amino acids, more preferably byfour amino acids, even more preferably by three amino acids, mostpreferably by two amino acids, and even most preferably by one aminoacid from the mature polypeptide of SEQ ID NO: 34.

In another aspect, the CEL6 polypeptide having cellobiohydrolase IIactivity comprises the amino acid sequence of SEQ ID NO: 34 or anallelic variant thereof; or a fragment thereof that hascellobiohydrolase activity. In a preferred aspect, the polypeptidecomprises the amino acid sequence of SEQ ID NO: 34. In another preferredaspect, the polypeptide comprises the mature polypeptide of SEQ ID NO:34. In another preferred aspect, the polypeptide comprises amino acids18 to 481 of SEQ ID NO: 34, or an allelic variant thereof; or a fragmentthereof that has cellobiohydrolase activity. In another preferredaspect, the polypeptide comprises amino acids 18 to 481 of SEQ ID NO:34. In another preferred aspect, the polypeptide consists of the aminoacid sequence of SEQ ID NO: 34 or an allelic variant thereof; or afragment thereof that has cellobiohydrolase activity. In anotherpreferred aspect, the polypeptide consists of the amino acid sequence ofSEQ ID NO: 34. In another preferred aspect, the polypeptide consists ofthe mature polypeptide of SEQ ID NO: 34. In another preferred aspect,the polypeptide consists of amino acids 18 to 481 of SEQ ID NO: 34 or anallelic variant thereof; or a fragment thereof that hascellobiohydrolase activity. In another preferred aspect, the polypeptideconsists of amino acids 18 to 481 of SEQ ID NO: 34.

In another aspect, the CEL6 polypeptide having cellobiohydrolase IIactivity is encoded by a polynucleotide that hybridizes under preferablyvery low stringency conditions, more preferably low stringencyconditions, more preferably medium stringency conditions, morepreferably medium-high stringency conditions, even more preferably highstringency conditions, and most preferably very high stringencyconditions with (i) the mature polypeptide coding sequence of SEQ ID NO:33, (ii) the genomic DNA sequence comprising the mature polypeptidecoding sequence of SEQ ID NO: 33, or (iii) a complementary strand of (i)or (ii) (J. Sambrook, E. F. Fritsch, and T. Maniatis, 1989, MolecularCloning, A Laboratory Manual, 2d edition, Cold Spring Harbor, N.Y.). Ina preferred aspect, the mature polypeptide coding sequence isnucleotides 52 to 1443 of SEQ ID NO: 33.

In another aspect, the CEL6 polypeptide having cellobiohydrolase IIactivity is encoded by a polynucleotide comprising or consisting of anucleotide sequence that has a degree of identity to the maturepolypeptide coding sequence of SEQ ID NO: 33 of preferably at least 60%,more preferably at least 65%, more preferably at least 70%, morepreferably at least 75%, more preferably at least 80%, more preferablyat least 85%, even more preferably at least 90%, most preferably atleast 95%, and even most preferably at least 96%, at least 97%, at least98%, or at least 99%, which encode an active polypeptide.

In another aspect, the CEL6 polypeptide having cellobiohydrolase IIactivity is encoded by a polynucleotide comprising or consisting of thenucleotide sequence of SEQ ID NO: 33. In another preferred aspect, thenucleotide sequence comprises or consists of the mature polypeptidecoding region of SEQ ID NO: 33. In another preferred aspect, thenucleotide sequence comprises or consists of nucleotides 52 to 1443 ofSEQ ID NO: 33. The present invention also encompasses nucleotidesequences which encode a polypeptide comprising or consisting of theamino acid sequence of SEQ ID NO: 34 or the mature polypeptide thereof,which differ from SEQ ID NO: 33 or the mature polypeptide codingsequence thereof by virtue of the degeneracy of the genetic code. Thepresent invention also relates to subsequences of SEQ ID NO: 33 whichencode fragments of SEQ ID NO: 34 that have cellobiohydrolase activity.

In another preferred aspect, the CEL6 polypeptide havingcellobiohydrolase II activity is obtained from Trichoderma reesei.

In one aspect, the CEL6 polypeptide having cellobiohydrolase II activitycomprises an amino acid sequence having a degree of identity to themature polypeptide of SEQ ID NO: 26 of preferably at least 60%, morepreferably at least 65%, more preferably at least 70%, more preferablyat least 75%, more preferably at least 80%, more preferably at least85%, even more preferably at least 90%, most preferably at least 95%,and even most preferably at least 96%, at least 97%, at least 98%, or atleast 99%, which have cellobiohydrolase activity (hereinafter“homologous polypeptides”). In another preferred aspect, the homologouspolypeptides comprise amino acid sequences which differ preferably byten amino acids, more preferably by five amino acids, more preferably byfour amino acids, even more preferably by three amino acids, mostpreferably by two amino acids, and even most preferably by one aminoacid from the mature polypeptide of SEQ ID NO: 26.

In another aspect, the CEL6 polypeptide having cellobiohydrolase IIactivity comprises the amino acid sequence of SEQ ID NO: 26 or anallelic variant thereof; or a fragment thereof that hascellobiohydrolase activity. In a preferred aspect, the polypeptidecomprises the amino acid sequence of SEQ ID NO: 26. In another preferredaspect, the polypeptide comprises the mature polypeptide of SEQ ID NO:26. In another preferred aspect, the polypeptide comprises amino acids25 to 471 of SEQ ID NO: 26, or an allelic variant thereof; or a fragmentthereof that has cellobiohydrolase activity. In another preferredaspect, the polypeptide comprises amino acids 25 to 471 of SEQ ID NO:26. In another preferred aspect, the polypeptide consists of the aminoacid sequence of SEQ ID NO: 26 or an allelic variant thereof; or afragment thereof that has cellobiohydrolase activity. In anotherpreferred aspect, the polypeptide consists of the amino acid sequence ofSEQ ID NO: 26. In another preferred aspect, the polypeptide consists ofthe mature polypeptide of SEQ ID NO: 26. In another preferred aspect,the polypeptide consists of amino acids 25 to 471 of SEQ ID NO: 26 or anallelic variant thereof; or a fragment thereof that hascellobiohydrolase activity. In another preferred aspect, the polypeptideconsists of amino acids 25 to 471 of SEQ ID NO: 26.

In another aspect, the CEL6 polypeptide having cellobiohydrolase IIactivity is encoded by a polynucleotide that hybridizes under preferablyvery low stringency conditions, more preferably low stringencyconditions, more preferably medium stringency conditions, morepreferably medium-high stringency conditions, even more preferably highstringency conditions, and most preferably very high stringencyconditions with (i) the mature polypeptide coding sequence of SEQ ID NO:25, (ii) the genomic DNA sequence comprising the mature polypeptidecoding sequence of SEQ ID NO: 25, or (iii) a complementary strand of (i)or (ii). In a preferred aspect, the mature polypeptide coding sequenceis nucleotides 125 to 1465 of SEQ ID NO: 25.

In another aspect, the CEL6 polypeptide having cellobiohydrolase IIactivity is encoded by a polynucleotide comprising or consisting of anucleotide sequence that has a degree of identity to the maturepolypeptide coding sequence of SEQ ID NO: 25 of preferably at least 60%,more preferably at least 65%, more preferably at least 70%, morepreferably at least 75%, more preferably at least 80%, more preferablyat least 85%, even more preferably at least 90%, most preferably atleast 95%, and even most preferably at least 96%, at least 97%, at least98%, or at least 99%, which encode an active polypeptide.

In another aspect, the CEL6 polypeptide having cellobiohydrolase IIactivity is encoded by a polynucleotide comprising or consisting of thenucleotide sequence of SEQ ID NO: 25. In another preferred aspect, thenucleotide sequence comprises or consists of the mature polypeptidecoding region of SEQ ID NO: 25. In another preferred aspect, thenucleotide sequence comprises or consists of nucleotides 125 to 1465 ofSEQ ID NO: 25. The present invention also encompasses nucleotidesequences which encode a polypeptide comprising or consisting of theamino acid sequence of SEQ ID NO: 26 or the mature polypeptide thereof,which differ from SEQ ID NO: 25 or the mature polypeptide codingsequence thereof by virtue of the degeneracy of the genetic code. Thepresent invention also relates to subsequences of SEQ ID NO: 25 whichencode fragments of SEQ ID NO: 26 that have cellobiohydrolase activity.

In another preferred aspect, the CEL6 polypeptide havingcellobiohydrolase II activity is obtained from Chaetomium thermophilum.

In one aspect, the CEL6 polypeptide having cellobiohydrolase II activitycomprises an amino acid sequence having a degree of identity to themature polypeptide of SEQ ID NO: 38 of preferably at least 60%, morepreferably at least 65%, more preferably at least 70%, more preferablyat least 75%, more preferably at least 80%, more preferably at least85%, even more preferably at least 90%, most preferably at least 95%,and even most preferably at least 96%, at least 97%, at least 98%, or atleast 99%, which have cellobiohydrolase II activity (hereinafter“homologous polypeptides”). In another preferred aspect, the homologouspolypeptides comprise amino acid sequences which differ preferably byten amino acids, more preferably by five amino acids, more preferably byfour amino acids, even more preferably by three amino acids, mostpreferably by two amino acids, and even most preferably by one aminoacid from the mature polypeptide of SEQ ID NO: 38.

In another aspect, the CEL6 polypeptide having cellobiohydrolase IIactivity comprises the amino acid sequence of SEQ ID NO: 38 or anallelic variant thereof; or a fragment thereof that hascellobiohydrolase II activity. In a preferred aspect, the polypeptidecomprises the amino acid sequence of SEQ ID NO: 38. In another preferredaspect, the polypeptide comprises the mature polypeptide of SEQ ID NO:38. In another preferred aspect, the polypeptide comprises amino acids18 to 477 of SEQ ID NO: 38, or an allelic variant thereof; or a fragmentthereof that has cellobiohydrolase II activity. In another preferredaspect, the polypeptide comprises amino acids 18 to 477 of SEQ ID NO:38. In another preferred aspect, the polypeptide consists of the aminoacid sequence of SEQ ID NO: 38 or an allelic variant thereof; or afragment thereof that has cellobiohydrolase II activity. In anotherpreferred aspect, the polypeptide consists of the amino acid sequence ofSEQ ID NO: 38. In another preferred aspect, the polypeptide consists ofthe mature polypeptide of SEQ ID NO: 38. In another preferred aspect,the polypeptide consists of amino acids 18 to 477 of SEQ ID NO: 38 or anallelic variant thereof; or a fragment thereof that hascellobiohydrolase II activity. In another preferred aspect, thepolypeptide consists of amino acids 18 to 477 of SEQ ID NO: 38.

In another aspect, the CEL6 polypeptide having cellobiohydrolase IIactivity is encoded by a polynucleotide that hybridizes under preferablyvery low stringency conditions, more preferably low stringencyconditions, more preferably medium stringency conditions, morepreferably medium-high stringency conditions, even more preferably highstringency conditions, and most preferably very high stringencyconditions with (i) the mature polypeptide coding sequence of SEQ ID NO:37, (ii) the genomic DNA sequence comprising the mature polypeptidecoding sequence of SEQ ID NO: 37, or (iii) a complementary strand of (i)or (ii). In a preferred aspect, the mature polypeptide coding sequenceis nucleotides 15 to 1731 of SEQ ID NO: 37.

In another aspect, the CEL6 polypeptide having cellobiohydrolase IIactivity is encoded by a polynucleotide comprising or consisting of anucleotide sequence that has a degree of identity to the maturepolypeptide coding sequence of SEQ ID NO: 37 of preferably at least 60%,more preferably at least 65%, more preferably at least 70%, morepreferably at least 75%, more preferably at least 80%, more preferablyat least 85%, even more preferably at least 90%, most preferably atleast 95%, and even most preferably at least 96%, at least 97%, at least98%, or at least 99%, which encode an active polypeptide.

In another aspect, the CEL6 polypeptide having cellobiohydrolase IIactivity is encoded by a polynucleotide comprising or consisting of thenucleotide sequence of SEQ ID NO: 37. In another preferred aspect, thenucleotide sequence comprises or consists of the mature polypeptidecoding region of SEQ ID NO: 37. In another preferred aspect, thenucleotide sequence comprises or consists of nucleotides 15 to 1731 ofSEQ ID NO: 37. The present invention also encompasses nucleotidesequences which encode a polypeptide comprising or consisting of theamino acid sequence of SEQ ID NO: 38 or the mature polypeptide thereof,which differ from SEQ ID NO: 37 or the mature polypeptide codingsequence thereof by virtue of the degeneracy of the genetic code. Thepresent invention also relates to subsequences of SEQ ID NO: 37 whichencode fragments of SEQ ID NO: 38 that have cellobiohydrolase IIactivity.

For purposes of the present invention, hybridization indicates that thenucleotide sequence hybridizes to a labeled nucleic acid probecorresponding to the mature polypeptide coding sequence or itsfull-length complementary strand; under very low to very high stringencyconditions. Molecules to which the nucleic acid probe hybridizes underthese conditions can be detected using, for example, X-ray film. Forlong probes of at least 100 nucleotides in length, very low to very highstringency conditions are defined as prehybridization and hybridizationat 42° C. in 5×SSPE, 0.3% SDS, 200 μg/ml sheared and denatured salmonsperm DNA, and either 25% formamide for very low and low stringencies,35% formamide for medium and medium-high stringencies, or 50% formamidefor high and very high stringencies, following standard Southernblotting procedures for 12 to 24 hours optimally. For long probes of atleast 100 nucleotides in length, the carrier material is finally washedthree times each for 15 minutes using 2×SSC, 0.2% SDS preferably atleast at 45° C. (very low stringency), more preferably at least at 50°C. (low stringency), more preferably at least at 55° C. (mediumstringency), more preferably at least at 60° C. (medium-highstringency), even more preferably at least at 65° C. (high stringency),and most preferably at least at 70° C. (very high stringency).

The techniques used to isolate or clone a polynucleotide encoding apolypeptide are known in the art and include isolation from genomic DNA,preparation from cDNA, or a combination thereof. The cloning of thepolynucleotides of the present invention from such genomic DNA can beeffected, e.g., by using the well known polymerase chain reaction (PCR)or antibody screening of expression libraries to detect cloned DNAfragments with shared structural features. See, e.g., Innis et al.,1990, PCR: A Guide to Methods and Application, Academic Press, New York.Other nucleic acid amplification procedures such as ligase chainreaction (LCR), ligated activated transcription (LAT) and nucleotidesequence-based amplification (NASBA) may be used. The polynucleotidesmay be cloned from a strain of Myceliophthora, or another or relatedorganism and thus, for example, may be an allelic or species variant ofthe polypeptide encoding region of the nucleotide sequence.

Polypeptides Having Xylanase Activity and Polynucleotides Thereof

In the methods of the present invention, the enzyme compositioncomprises a polypeptide having xylanase activity. The polypeptide havingxylanase activity may be obtained from microorganisms of any genus. In apreferred aspect, the polypeptide obtained from a given source issecreted extracellularly.

A polypeptide having xylanase activity may be a bacterial polypeptide.For example, the polypeptide may be a gram positive bacterialpolypeptide such as a Bacillus, Streptococcus, Streptomyces,Staphylococcus, Enterococcus, Lactobacillus, Lactococcus, Clostridium,Geobacillus, or Oceanobacillus polypeptide having xylanase activity, ora Gram negative bacterial polypeptide such as an E. coli, Pseudomonas,Salmonella, Campylobacter, Helicobacter, Flavobacterium, Fusobacterium,Ilyobacter, Neisseria, or Ureaplasma polypeptide having xylanaseactivity.

In a preferred aspect, the polypeptide is a Bacillus alkalophilus,Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans,Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus,Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacilluspumilus, Bacillus stearothermophilus, Bacillus subtilis, or Bacillusthuringiensis polypeptide having xylanase activity.

In another preferred aspect, the polypeptide is a Streptococcusequisimilis, Streptococcus pyogenes, Streptococcus uberis, orStreptococcus equi subsp. Zooepidemicus polypeptide having xylanaseactivity.

In another preferred aspect, the polypeptide is a Streptomycesachromogenes, Streptomyces avermitilis, Streptomyces coelicolor,Streptomyces griseus, or Streptomyces lividans polypeptide havingxylanase activity.

The polypeptide having xylanase activity may also be a fungalpolypeptide, and more preferably a yeast polypeptide such as a Candida,Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowiapolypeptide having xylanase activity; or more preferably a filamentousfungal polypeptide such as an Acremonium, Agaricus, Alternaria,Aspergillus, Aureobasidium, Botryospaeria, Ceriporiopsis, Chaetomidium,Chrysosporium, Claviceps, Cochliobolus, Coprinopsis, Coptotermes,Corynascus, Cryphonectria, Cryptococcus, Diplodia, Exidia, Filibasidium,Fusarium, Gibberella, Holomastigotoides, Humicola, Irpex, Lentinula,Leptospaeria, Magnaporthe, Melanocarpus, Meripilus, Mucor,Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium,Phanerochaete, Piromyces, Poitrasia, Pseudoplectania,Pseudotrichonympha, Rhizomucor, Schizophyllum, Scytalidium, Talaromyces,Thermoascus, Thielavia, Tolypocladium, Trichoderma, Trichophaea,Verticillium, Volvariella, or Xylaria polypeptide having xylanaseactivity.

In a preferred aspect, the polypeptide is a Saccharomycescarlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus,Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomycesnorbensis, or Saccharomyces oviformis polypeptide having xylanaseactivity.

In another preferred aspect, the polypeptide is an Acremoniumcellulolyticus, Aspergillus aculeatus, Aspergillus awamori, Aspergillusfumigatus, Aspergillus foetidus, Aspergillus japonicus, Aspergillusnidulans, Aspergillus niger, Aspergillus oryzae, Chrysosporiumkeratinophilum, Chrysosporium lucknowense, Chrysosporium tropicum,Chrysosporium merdarium, Chrysosporium inops, Chrysosporium pannicola,Chrysosporium queenslandicum, Chrysosporium zonatum, Fusariumbactridioides, Fusarium cerealis, Fusarium crookwellense, Fusariumculmorum, Fusarium graminearum, Fusarium graminum, Fusariumheterosporum, Fusarium negundi, Fusarium oxysporum, Fusariumreticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum,Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum,Fusarium trichothecioides, Fusarium venenatum, Humicola grisea, Humicolainsolens, Humicola lanuginosa, Irpex lacteus, Mucor miehei,Myceliophthora thermophila, Neurospora crassa, Penicillium funiculosum,Penicillium purpurogenum, Phanerochaete chtysosporium, Thielaviaachromatica, Thielavia albomyces, Thielavia albopilosa, Thielaviaaustraleinsis, Thielavia fimeti, Thielavia microspora, Thielaviaovispora, Thielavia peruviana, Thielavia spededonium, Thielavia setosa,Thielavia subthermophila, Thielavia terrestris, Trichoderma harzianum,Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei,Trichoderma viride, or Trichophaea saccata polypeptide having xylanaseactivity.

In a preferred aspect, the polypeptide having xylanase activity is aGH10 polypeptide. In another preferred aspect, the polypeptide havingxylanase activity is a GH11 polypeptide.

In another preferred aspect, the GH10 polypeptide having xylanaseactivity is obtained from Aspergillus aculeatus.

In one aspect, the GH10 polypeptide having xylanase activity comprisesan amino acid sequence having a degree of identity to the maturepolypeptide of SEQ ID NO: 70 of preferably at least 60%, more preferablyat least 65%, more preferably at least 70%, more preferably at least75%, more preferably at least 80%, more preferably at least 85%, evenmore preferably at least 90%, most preferably at least 95%, and evenmost preferably at least 96%, at least 97%, at least 98%, or at least99%, which have xylanase activity (hereinafter “homologouspolypeptides”). In another preferred aspect, the homologous polypeptidescomprise amino acid sequences which differ preferably by ten aminoacids, more preferably by five amino acids, more preferably by fouramino acids, even more preferably by three amino acids, most preferablyby two amino acids, and even most preferably by one amino acid from themature polypeptide of SEQ ID NO: 70.

In another aspect, the GH10 polypeptide having xylanase activitycomprises the amino acid sequence of SEQ ID NO: 70 or an allelic variantthereof; or a fragment thereof that has xylanase activity. In apreferred aspect, the polypeptide comprises the amino acid sequence ofSEQ ID NO: 70. In another preferred aspect, the polypeptide comprisesthe mature polypeptide of SEQ ID NO: 70. In another preferred aspect,the polypeptide comprises amino acids 23 to 406 of SEQ ID NO: 70, or anallelic variant thereof; or a fragment thereof that has xylanaseactivity. In another preferred aspect, the polypeptide comprises aminoacids 23 to 406 of SEQ ID NO: 70. In another preferred aspect, thepolypeptide consists of the amino acid sequence of SEQ ID NO: 70 or anallelic variant thereof; or a fragment thereof that has xylanaseactivity. In another preferred aspect, the polypeptide consists of theamino acid sequence of SEQ ID NO: 70. In another preferred aspect, thepolypeptide consists of the mature polypeptide of SEQ ID NO: 70. Inanother preferred aspect, the polypeptide consists of amino acids 23 to406 of SEQ ID NO: 70 or an allelic variant thereof; or a fragmentthereof that has xylanase activity. In another preferred aspect, thepolypeptide consists of amino acids 23 to 406 of SEQ ID NO: 70.

In another aspect, the GH10 polypeptide having xylanase activity isencoded by a polynucleotide that hybridizes under preferably very lowstringency conditions, more preferably low stringency conditions, morepreferably medium stringency conditions, more preferably medium-highstringency conditions, even more preferably high stringency conditions,and most preferably very high stringency conditions with (i) the maturepolypeptide coding sequence of SEQ ID NO: 69, (ii) the genomic DNAsequence comprising the mature polypeptide coding sequence of SEQ ID NO:69, or (iii) a complementary strand of (i) or (ii). In a preferredaspect, the mature polypeptide coding sequence is nucleotides 69 to 1314of SEQ ID NO: 69.

In another aspect, the GH10 polypeptide having xylanase activity isencoded by a polynucleotide comprising or consisting of a nucleotidesequence that has a degree of identity to the mature polypeptide codingsequence of SEQ ID NO: 69 of preferably at least 60%, more preferably atleast 65%, more preferably at least 70%, more preferably at least 75%,more preferably at least 80%, more preferably at least 85%, even morepreferably at least 90%, most preferably at least 95%, and even mostpreferably at least 96%, at least 97%, at least 98%, or at least 99%,which encode an active polypeptide.

In another aspect, the GH10 polypeptide having xylanase activity isencoded by a polynucleotide comprising or consisting of the nucleotidesequence of SEQ ID NO: 69. In another preferred aspect, the nucleotidesequence comprises or consists of the mature polypeptide coding regionof SEQ ID NO: 69. In another preferred aspect, the nucleotide sequencecomprises or consists of nucleotides 69 to 1314 of SEQ ID NO: 69. Thepresent invention also encompasses nucleotide sequences which encode thepolypeptide comprising or consisting of the amino acid sequence of SEQID NO: 70 or the mature polypeptide thereof, which differ from SEQ IDNO: 69 or the mature polypeptide coding sequence thereof by virtue ofthe degeneracy of the genetic code. The present invention also relatesto subsequences of SEQ ID NO: 69 which encode fragments of SEQ ID NO: 70that have xylanase activity.

In another preferred aspect, the GH10 polypeptide having xylanaseactivity is obtained from Thielavia terrestris NRRL 8126.

In one aspect, the GH10 polypeptide having xylanase activity comprisesan amino acid sequence having a degree of identity to the maturepolypeptide of SEQ ID NO: 72 of preferably at least 60%, more preferablyat least 65%, more preferably at least 70%, more preferably at least75%, more preferably at least 80%, more preferably at least 85%, evenmore preferably at least 90%, most preferably at least 95%, and evenmost preferably at least 96%, at least 97%, at least 98%, or at least99%, which have xylanase activity (hereinafter “homologouspolypeptides”). In another preferred aspect, the homologous polypeptidescomprise amino acid sequences which differ preferably by ten aminoacids, more preferably by five amino acids, more preferably by fouramino acids, even more preferably by three amino acids, most preferablyby two amino acids, and even most preferably by one amino acid from themature polypeptide of SEQ ID NO: 72.

In another aspect, the GH10 polypeptide having xylanase activitycomprises the amino acid sequence of SEQ ID NO: 72 or an allelic variantthereof; or a fragment thereof that has xylanase activity. In apreferred aspect, the polypeptide comprises the amino acid sequence ofSEQ ID NO: 72. In another preferred aspect, the polypeptide comprisesthe mature polypeptide of SEQ ID NO: 72. In another preferred aspect,the polypeptide comprises amino acids 20 to 369 of SEQ ID NO: 72, or anallelic variant thereof; or a fragment thereof that has xylanaseactivity. In another preferred aspect, the polypeptide comprises aminoacids 20 to 369 of SEQ ID NO: 72. In another preferred aspect, thepolypeptide consists of the amino acid sequence of SEQ ID NO: 72 or anallelic variant thereof; or a fragment thereof that has xylanaseactivity. In another preferred aspect, the polypeptide consists of theamino acid sequence of SEQ ID NO: 72. In another preferred aspect, thepolypeptide consists of the mature polypeptide of SEQ ID NO: 72. Inanother preferred aspect, the polypeptide consists of amino acids 20 to369 of SEQ ID NO: 72 or an allelic variant thereof; or a fragmentthereof that has xylanase activity. In another preferred aspect, thepolypeptide consists of amino acids 20 to 369 of SEQ ID NO: 72.

In another aspect, the GH10 polypeptide having xylanase activity isencoded by a polynucleotide that hybridizes under preferably very lowstringency conditions, more preferably low stringency conditions, morepreferably medium stringency conditions, more preferably medium-highstringency conditions, even more preferably high stringency conditions,and most preferably very high stringency conditions with (i) the maturepolypeptide coding sequence of SEQ ID NO: 71, (ii) the genomic DNAsequence comprising the mature polypeptide coding sequence of SEQ ID NO:71, or (iii) a complementary strand of (i) or (ii). In a preferredaspect, the mature polypeptide coding sequence is nucleotides 58 to 1107of SEQ ID NO: 71.

In another aspect, the GH10 polypeptide having xylanase activity isencoded by a polynucleotide comprising or consisting of a nucleotidesequence that has a degree of identity to the mature polypeptide codingsequence of SEQ ID NO: 71 of preferably at least 60%, more preferably atleast 65%, more preferably at least 70%, more preferably at least 75%,more preferably at least 80%, more preferably at least 85%, even morepreferably at least 90%, most preferably at least 95%, and even mostpreferably at least 96%, at least 97%, at least 98%, or at least 99%,which encode an active polypeptide.

In another aspect, the GH10 polypeptide having xylanase activity isencoded by a polynucleotide comprising or consisting of the nucleotidesequence of SEQ ID NO: 71. In another preferred aspect, the nucleotidesequence comprises or consists of the mature polypeptide coding regionof SEQ ID NO: 71. In another preferred aspect, the nucleotide sequencecomprises or consists of nucleotides 58 to 1107 of SEQ ID NO: 71. Thepresent invention also encompasses nucleotide sequences which encode apolypeptide comprising or consisting of the amino acid sequence of SEQID NO: 72 or the mature polypeptide thereof, which differ from SEQ IDNO: 71 or the mature polypeptide coding sequence thereof by virtue ofthe degeneracy of the genetic code. The present invention also relatesto subsequences of SEQ ID NO: 71 which encode fragments of SEQ ID NO: 72that have xylanase activity.

In another aspect, the GH10 polypeptide having xylanase activitycomprises an amino acid sequence having a degree of identity to themature polypeptide of SEQ ID NO: 74 of preferably at least 60%, morepreferably at least 65%, more preferably at least 70%, more preferablyat least 75%, more preferably at least 80%, more preferably at least85%, even more preferably at least 90%, most preferably at least 95%,and even most preferably at least 96%, at least 97%, at least 98%, or atleast 99%, which have xylanase activity (hereinafter “homologouspolypeptides”). In another preferred aspect, the homologous polypeptidescomprise amino acid sequences which differ preferably by ten aminoacids, more preferably by five amino acids, more preferably by fouramino acids, even more preferably by three amino acids, most preferablyby two amino acids, and even most preferably by one amino acid from themature polypeptide of SEQ ID NO: 74.

In another aspect, the GH10 polypeptide having xylanase activitycomprises the amino acid sequence of SEQ ID NO: 74 or an allelic variantthereof; or a fragment thereof that has xylanase activity. In apreferred aspect, the polypeptide comprises the amino acid sequence ofSEQ ID NO: 74. In another preferred aspect, the polypeptide comprisesthe mature polypeptide of SEQ ID NO: 74. In another preferred aspect,the polypeptide comprises amino acids 19 to 414 of SEQ ID NO: 74, or anallelic variant thereof; or a fragment thereof that has xylanaseactivity. In another preferred aspect, the polypeptide comprises aminoacids 19 to 414 of SEQ ID NO: 74. In another preferred aspect, thepolypeptide consists of the amino acid sequence of SEQ ID NO: 74 or anallelic variant thereof; or a fragment thereof that has xylanaseactivity. In another preferred aspect, the polypeptide consists of theamino acid sequence of SEQ ID NO: 74. In another preferred aspect, thepolypeptide consists of the mature polypeptide of SEQ ID NO: 74. Inanother preferred aspect, the polypeptide consists of amino acids 19 to414 of SEQ ID NO: 74 or an allelic variant thereof; or a fragmentthereof that has xylanase activity. In another preferred aspect, thepolypeptide consists of amino acids 19 to 414 of SEQ ID NO: 74.

In another aspect, the GH10 polypeptide having xylanase activity isencoded by a polynucleotide that hybridizes under preferably very lowstringency conditions, more preferably low stringency conditions, morepreferably medium stringency conditions, more preferably medium-highstringency conditions, even more preferably high stringency conditions,and most preferably very high stringency conditions with (i) the maturepolypeptide coding sequence of SEQ ID NO: 73, (ii) the genomic DNAsequence comprising the mature polypeptide coding sequence of SEQ ID NO:73, or (iii) a complementary strand of (i) or (ii). In a preferredaspect, the mature polypeptide coding sequence is nucleotides 55 to 1242of SEQ ID NO: 73.

In another aspect, the GH10 polypeptide having xylanase activity isencoded by a polynucleotide comprising or consisting of a nucleotidesequence that has a degree of identity to the mature polypeptide codingsequence of SEQ ID NO: 73 of preferably at least 60%, more preferably atleast 65%, more preferably at least 70%, more preferably at least 75%,more preferably at least 80%, more preferably at least 85%, even morepreferably at least 90%, most preferably at least 95%, and even mostpreferably at least 96%, at least 97%, at least 98%, or at least 99%,which encode an active polypeptide.

In another aspect, the GH10 polypeptide having xylanase activity isencoded by a polynucleotide comprising or consisting of the nucleotidesequence of SEQ ID NO: 73. In another preferred aspect, the nucleotidesequence comprises or consists of the mature polypeptide coding regionof SEQ ID NO: 73. In another preferred aspect, the nucleotide sequencecomprises or consists of nucleotides 55 to 1242 of SEQ ID NO: 73. Thepresent invention also encompasses nucleotide sequences which encode apolypeptide comprising or consisting of the amino acid sequence of SEQID NO: 74 or the mature polypeptide thereof, which differ from SEQ IDNO: 73 or the mature polypeptide coding sequence thereof by virtue ofthe degeneracy of the genetic code. The present invention also relatesto subsequences of SEQ ID NO: 73 which encode fragments of SEQ ID NO: 74that have xylanase activity.

In another preferred aspect, the GH10 polypeptide having xylanaseactivity is obtained from Aspergillus fumigatus.

In another aspect, the GH10 polypeptide having xylanase activitycomprises an amino acid sequence having a degree of identity to themature polypeptide of SEQ ID NO: 76 of preferably at least 60%, morepreferably at least 65%, more preferably at least 70%, more preferablyat least 75%, more preferably at least 80%, more preferably at least85%, even more preferably at least 90%, most preferably at least 95%,and even most preferably at least 96%, at least 97%, at least 98%, or atleast 99%, which have xylanase activity (hereinafter “homologouspolypeptides”). In another preferred aspect, the homologous polypeptidescomprise amino acid sequences which differ preferably by ten aminoacids, more preferably by five amino acids, more preferably by fouramino acids, even more preferably by three amino acids, most preferablyby two amino acids, and even most preferably by one amino acid from themature polypeptide of SEQ ID NO: 76.

In another aspect, the GH10 polypeptide having xylanase activitycomprises the amino acid sequence of SEQ ID NO: 76 or an allelic variantthereof; or a fragment thereof that has xylanase activity. In apreferred aspect, the polypeptide comprises the amino acid sequence ofSEQ ID NO: 76. In another preferred aspect, the polypeptide comprisesthe mature polypeptide of SEQ ID NO: 76. In another preferred aspect,the polypeptide comprises amino acids 18 to 364 of SEQ ID NO: 76, or anallelic variant thereof; or a fragment thereof that has xylanaseactivity. In another preferred aspect, the polypeptide comprises aminoacids 18 to 364 of SEQ ID NO: 76. In another preferred aspect, thepolypeptide consists of the amino acid sequence of SEQ ID NO: 76 or anallelic variant thereof; or a fragment thereof that has xylanaseactivity. In another preferred aspect, the polypeptide consists of theamino acid sequence of SEQ ID NO: 76. In another preferred aspect, thepolypeptide consists of the mature polypeptide of SEQ ID NO: 76. Inanother preferred aspect, the polypeptide consists of amino acids 18 to364 of SEQ ID NO: 76 or an allelic variant thereof; or a fragmentthereof that has xylanase activity. In another preferred aspect, thepolypeptide consists of amino acids 18 to 364 of SEQ ID NO: 76.

In another aspect, the GH10 polypeptide having xylanase activity isencoded by a polynucleotide that hybridizes under preferably very lowstringency conditions, more preferably low stringency conditions, morepreferably medium stringency conditions, more preferably medium-highstringency conditions, even more preferably high stringency conditions,and most preferably very high stringency conditions with (i) the maturepolypeptide coding sequence of SEQ ID NO: 75, (ii) the genomic DNAsequence comprising the mature polypeptide coding sequence of SEQ ID NO:75, or (iii) a complementary strand of (i) or (ii). In a preferredaspect, the mature polypeptide coding sequence is nucleotides 52 to 1145of SEQ ID NO: 75.

In another aspect, the GH10 polypeptide having xylanase activity isencoded by a polynucleotide comprising or consisting of a nucleotidesequence that has a degree of identity to the mature polypeptide codingsequence of SEQ ID NO: 75 of preferably at least 60%, more preferably atleast 65%, more preferably at least 70%, more preferably at least 75%,more preferably at least 80%, more preferably at least 85%, even morepreferably at least 90%, most preferably at least 95%, and even mostpreferably at least 96%, at least 97%, at least 98%, or at least 99%,which encode an active polypeptide.

In another aspect, the GH10 polypeptide having xylanase activity isencoded by a polynucleotide comprising or consisting of the nucleotidesequence of SEQ ID NO: 75. In another preferred aspect, the nucleotidesequence comprises or consists of the mature polypeptide coding regionof SEQ ID NO: 75. In another preferred aspect, the nucleotide sequencecomprises or consists of nucleotides 52 to 1145 of SEQ ID NO: 75. Thepresent invention also encompasses nucleotide sequences which encode apolypeptide comprising or consisting of the amino acid sequence of SEQID NO: 76 or the mature polypeptide thereof, which differ from SEQ IDNO: 75 or the mature polypeptide coding sequence thereof by virtue ofthe degeneracy of the genetic code. The present invention also relatesto subsequences of SEQ ID NO: 75 which encode fragments of SEQ ID NO: 76that have xylanase activity.

In another aspect, the GH10 polypeptide having xylanase activitycomprises an amino acid sequence having a degree of identity to themature polypeptide of SEQ ID NO: 78 of preferably at least 60%, morepreferably at least 65%, more preferably at least 70%, more preferablyat least 75%, more preferably at least 80%, more preferably at least85%, even more preferably at least 90%, most preferably at least 95%,and even most preferably at least 96%, at least 97%, at least 98%, or atleast 99%, which have xylanase activity (hereinafter “homologouspolypeptides”). In another preferred aspect, the homologous polypeptidescomprise amino acid sequences which differ preferably by ten aminoacids, more preferably by five amino acids, more preferably by fouramino acids, even more preferably by three amino acids, most preferablyby two amino acids, and even most preferably by one amino acid from themature polypeptide of SEQ ID NO: 78.

In another aspect, the GH10 polypeptide having xylanase activitycomprises the amino acid sequence of SEQ ID NO: 78 or an allelic variantthereof; or a fragment thereof that has xylanase activity. In apreferred aspect, the polypeptide comprises the amino acid sequence ofSEQ ID NO: 78. In another preferred aspect, the polypeptide comprisesthe mature polypeptide of SEQ ID NO: 78. In another preferred aspect,the polypeptide comprises amino acids 20 to 323 of SEQ ID NO: 78, or anallelic variant thereof; or a fragment thereof that has xylanaseactivity. In another preferred aspect, the polypeptide comprises aminoacids 20 to 323 of SEQ ID NO: 78. In another preferred aspect, thepolypeptide consists of the amino acid sequence of SEQ ID NO: 78 or anallelic variant thereof; or a fragment thereof that has xylanaseactivity. In another preferred aspect, the polypeptide consists of theamino acid sequence of SEQ ID NO: 78. In another preferred aspect, thepolypeptide consists of the mature polypeptide of SEQ ID NO: 78. Inanother preferred aspect, the polypeptide consists of amino acids 20 to323 of SEQ ID NO: 78 or an allelic variant thereof; or a fragmentthereof that has xylanase activity. In another preferred aspect, thepolypeptide consists of amino acids 20 to 323 of SEQ ID NO: 78.

In another aspect, the GH10 polypeptide having xylanase activity isencoded by a polynucleotide that hybridizes under preferably very lowstringency conditions, more preferably low stringency conditions, morepreferably medium stringency conditions, more preferably medium-highstringency conditions, even more preferably high stringency conditions,and most preferably very high stringency conditions with (i) the maturepolypeptide coding sequence of SEQ ID NO: 77, (ii) the genomic DNAsequence comprising the mature polypeptide coding sequence of SEQ ID NO:77, or (iii) a complementary strand of (i) or (ii). In a preferredaspect, the mature polypeptide coding sequence is nucleotides 58 to 1400of SEQ ID NO: 77.

In another aspect, the GH10 polypeptide having xylanase activity isencoded by a polynucleotide comprising or consisting of a nucleotidesequence that has a degree of identity to the mature polypeptide codingsequence of SEQ ID NO: 77 of preferably at least 60%, more preferably atleast 65%, more preferably at least 70%, more preferably at least 75%,more preferably at least 80%, more preferably at least 85%, even morepreferably at least 90%, most preferably at least 95%, and even mostpreferably at least 96%, at least 97%, at least 98%, or at least 99%,which encode an active polypeptide.

In another aspect, the GH10 polypeptide having xylanase activity isencoded by a polynucleotide comprising or consisting of the nucleotidesequence of SEQ ID NO: 77. In another preferred aspect, the nucleotidesequence comprises or consists of the mature polypeptide coding regionof SEQ ID NO: 77. In another preferred aspect, the nucleotide sequencecomprises or consists of nucleotides 58 to 1400 of SEQ ID NO: 77. Thepresent invention also encompasses nucleotide sequences which encode apolypeptide comprising or consisting of the amino acid sequence of SEQID NO: 78 or the mature polypeptide thereof, which differ from SEQ IDNO: 77 or the mature polypeptide coding sequence thereof by virtue ofthe degeneracy of the genetic code. The present invention also relatesto subsequences of SEQ ID NO: 77 which encode fragments of SEQ ID NO: 78that have xylanase activity.

In another aspect, the GH10 polypeptide having xylanase activitycomprises an amino acid sequence having a degree of identity to themature polypeptide of SEQ ID NO: 80 of preferably at least 60%, morepreferably at least 65%, more preferably at least 70%, more preferablyat least 75%, more preferably at least 80%, more preferably at least85%, even more preferably at least 90%, most preferably at least 95%,and even most preferably at least 96%, at least 97%, at least 98%, or atleast 99%, which have xylanase activity (hereinafter “homologouspolypeptides”). In another preferred aspect, the homologous polypeptidescomprise amino acid sequences which differ preferably by ten aminoacids, more preferably by five amino acids, more preferably by fouramino acids, even more preferably by three amino acids, most preferablyby two amino acids, and even most preferably by one amino acid from themature polypeptide of SEQ ID NO: 80.

In another aspect, the GH10 polypeptide having xylanase activitycomprises the amino acid sequence of SEQ ID NO: 80 or an allelic variantthereof; or a fragment thereof that has xylanase activity. In apreferred aspect, the polypeptide comprises the amino acid sequence ofSEQ ID NO: 80. In another preferred aspect, the polypeptide comprisesthe mature polypeptide of SEQ ID NO: 80. In another preferred aspect,the polypeptide comprises amino acids 20 to 397 of SEQ ID NO: 80, or anallelic variant thereof; or a fragment thereof that has xylanaseactivity. In another preferred aspect, the polypeptide comprises aminoacids 20 to 397 of SEQ ID NO: 80. In another preferred aspect, thepolypeptide consists of the amino acid sequence of SEQ ID NO: 80 or anallelic variant thereof; or a fragment thereof that has xylanaseactivity. In another preferred aspect, the polypeptide consists of theamino acid sequence of SEQ ID NO: 80. In another preferred aspect, thepolypeptide consists of the mature polypeptide of SEQ ID NO: 80. Inanother preferred aspect, the polypeptide consists of amino acids 20 to397 of SEQ ID NO: 80 or an allelic variant thereof; or a fragmentthereof that has xylanase activity. In another preferred aspect, thepolypeptide consists of amino acids 20 to 397 of SEQ ID NO: 80.

In another aspect, the GH10 polypeptide having xylanase activity isencoded by a polynucleotide that hybridizes under preferably very lowstringency conditions, more preferably low stringency conditions, morepreferably medium stringency conditions, more preferably medium-highstringency conditions, even more preferably high stringency conditions,and most preferably very high stringency conditions with (i) the maturepolypeptide coding sequence of SEQ ID NO: 79, (ii) the genomic DNAsequence comprising the mature polypeptide coding sequence of SEQ ID NO:79, or (iii) a complementary strand of (i) or (ii). In a preferredaspect, the mature polypeptide coding sequence is nucleotides 107 to1415 of SEQ ID NO: 79.

In another aspect, the GH10 polypeptide having xylanase activity isencoded by a polynucleotide comprising or consisting of a nucleotidesequence that has a degree of identity to the mature polypeptide codingsequence of SEQ ID NO: 79 of preferably at least 60%, more preferably atleast 65%, more preferably at least 70%, more preferably at least 75%,more preferably at least 80%, more preferably at least 85%, even morepreferably at least 90%, most preferably at least 95%, and even mostpreferably at least 96%, at least 97%, at least 98%, or at least 99%,which encode an active polypeptide.

In another aspect, the GH10 polypeptide having xylanase activity isencoded by a polynucleotide comprising or consisting of the nucleotidesequence of SEQ ID NO: 79. In another preferred aspect, the nucleotidesequence comprises or consists of the mature polypeptide coding regionof SEQ ID NO: 79. In another preferred aspect, the nucleotide sequencecomprises or consists of nucleotides 107 to 1415 of SEQ ID NO: 79. Thepresent invention also encompasses nucleotide sequences which encode apolypeptide comprising or consisting of the amino acid sequence of SEQID NO: 80 or the mature polypeptide thereof, which differ from SEQ IDNO: 79 or the mature polypeptide coding sequence thereof by virtue ofthe degeneracy of the genetic code. The present invention also relatesto subsequences of SEQ ID NO: 79 which encode fragments of SEQ ID NO: 80that have xylanase activity.

In another preferred aspect, the GH10 polypeptide having xylanaseactivity is obtained from Penicillium sp.

In another aspect, the GH10 polypeptide having xylanase activitycomprises an amino acid sequence having a degree of identity to themature polypeptide of SEQ ID NO: 99 of preferably at least 60%, morepreferably at least 65%, more preferably at least 70%, more preferablyat least 75%, more preferably at least 80%, more preferably at least85%, even more preferably at least 90%, most preferably at least 95%,and even most preferably at least 96%, at least 97%, at least 98%, or atleast 99%, which have xylanase activity (hereinafter “homologouspolypeptides”). In another preferred aspect, the homologous polypeptidescomprise amino acid sequences which differ preferably by ten aminoacids, more preferably by five amino acids, more preferably by fouramino acids, even more preferably by three amino acids, most preferablyby two amino acids, and even most preferably by one amino acid from themature polypeptide of SEQ ID NO: 99.

In another aspect, the GH10 polypeptide having xylanase activitycomprises the amino acid sequence of SEQ ID NO: 99 or an allelic variantthereof; or a fragment thereof that has xylanase activity. In apreferred aspect, the polypeptide comprises the amino acid sequence ofSEQ ID NO: 99. In another preferred aspect, the polypeptide comprisesthe mature polypeptide of SEQ ID NO: 99. In another preferred aspect,the polypeptide comprises amino acids 24 to 403 of SEQ ID NO: 99, or anallelic variant thereof; or a fragment thereof that has xylanaseactivity. In another preferred aspect, the polypeptide comprises aminoacids 24 to 403 of SEQ ID NO: 99. In another preferred aspect, thepolypeptide consists of the amino acid sequence of SEQ ID NO: 99 or anallelic variant thereof; or a fragment thereof that has xylanaseactivity. In another preferred aspect, the polypeptide consists of theamino acid sequence of SEQ ID NO: 99. In another preferred aspect, thepolypeptide consists of the mature polypeptide of SEQ ID NO: 99. Inanother preferred aspect, the polypeptide consists of amino acids 24 to403 of SEQ ID NO: 99 or an allelic variant thereof; or a fragmentthereof that has xylanase activity. In another preferred aspect, thepolypeptide consists of amino acids 24 to 403 of SEQ ID NO: 99.

In another aspect, the GH10 polypeptide having xylanase activity isencoded by a polynucleotide that hybridizes under preferably very lowstringency conditions, more preferably low stringency conditions, morepreferably medium stringency conditions, more preferably medium-highstringency conditions, even more preferably high stringency conditions,and most preferably very high stringency conditions with (i) the maturepolypeptide coding sequence of SEQ ID NO: 98, (ii) the cDNA sequencecontained in the mature polypeptide coding sequence of SEQ ID NO: 98, or(iii) a complementary strand of (i) or (ii). In a preferred aspect, themature polypeptide coding sequence is nucleotides 70 to 1385 of SEQ IDNO: 98.

In another aspect, the GH10 polypeptide having xylanase activity isencoded by a polynucleotide comprising or consisting of a nucleotidesequence that has a degree of identity to the mature polypeptide codingsequence of SEQ ID NO: 98 of preferably at least 60%, more preferably atleast 65%, more preferably at least 70%, more preferably at least 75%,more preferably at least 80%, more preferably at least 85%, even morepreferably at least 90%, most preferably at least 95%, and even mostpreferably at least 96%, at least 97%, at least 98%, or at least 99%,which encode an active polypeptide.

In another aspect, the GH10 polypeptide having xylanase activity isencoded by a polynucleotide comprising or consisting of the nucleotidesequence of SEQ ID NO: 98. In another preferred aspect, the nucleotidesequence comprises or consists of the mature polypeptide coding regionof SEQ ID NO: 98. In another preferred aspect, the nucleotide sequencecomprises or consists of nucleotides 70 to 1385 of SEQ ID NO: 98. Thepresent invention also encompasses nucleotide sequences which encode thepolypeptide comprising or consisting of the amino acid sequence of SEQID NO: 99 or the mature polypeptide thereof, which differ from SEQ IDNO: 98 or the mature polypeptide coding sequence thereof by virtue ofthe degeneracy of the genetic code. The present invention also relatesto subsequences of SEQ ID NO: 98 which encode fragments of SEQ ID NO: 99that have xylanase activity.

Nucleic Acid Constructs

An isolated polynucleotide encoding a cellulolytic protein, apolypeptide having cellulolytic enhancing activity, a polypeptide havingxylanase activity, or a polypeptide having cellobiohydrolase II activitymay be manipulated in a variety of ways to provide for expression of thepolypeptide by constructing a nucleic acid construct comprising anisolated polynucleotide encoding the polypeptide operably linked to oneor more (several) control sequences that direct the expression of thecoding sequence in a suitable host cell under conditions compatible withthe control sequences. Manipulation of the polynucleotide's sequenceprior to its insertion into a vector may be desirable or necessarydepending on the expression vector. The techniques for modifyingpolynucleotide sequences utilizing recombinant DNA methods are wellknown in the art.

The control sequence may be an appropriate promoter sequence, anucleotide sequence that is recognized by a host cell for expression ofa polynucleotide encoding such a polypeptide. The promoter sequencecontains transcriptional control sequences that mediate the expressionof the polypeptide. The promoter may be any nucleotide sequence thatshows transcriptional activity in the host cell of choice includingmutant, truncated, and hybrid promoters, and may be obtained from genesencoding extracellular or intracellular polypeptides either homologousor heterologous to the host cell.

Examples of suitable promoters for directing the transcription of thenucleic acid constructs, especially in a bacterial host cell, are thepromoters obtained from the E. coli lac operon, Streptomyces coelicoloragarase gene (dagA), Bacillus subtilis levansucrase gene (sacB),Bacillus licheniformis alpha-amylase gene (amyL), Bacillusstearothermophilus maltogenic amylase gene (amyM), Bacillusamyloliquefaciens alpha-amylase gene (amyQ), Bacillus licheniformispenicillinase gene (penP), Bacillus subtilis xylA and xylB genes, andprokaryotic beta-lactamase gene (Villa-Kamaroff et al., 1978,Proceedings of the National Academy of Sciences USA 75: 3727-3731), aswell as the tac promoter (DeBoer et al., 1983, Proceedings of theNational Academy of Sciences USA 80: 21-25). Further promoters aredescribed in “Useful proteins from recombinant bacteria” in ScientificAmerican, 1980, 242: 74-94; and in Sambrook et al., 1989, supra.

Examples of suitable promoters for directing the transcription of thenucleic acid constructs in a filamentous fungal host cell are promotersobtained from the genes for Aspergillus oryzae TAKA amylase, Rhizomucormiehei aspartic proteinase, Aspergillus niger neutral alpha-amylase,Aspergillus niger acid stable alpha-amylase, Aspergillus niger orAspergillus awamori glucoamylase (glaA), Rhizomucor miehei lipase,Aspergillus oryzae alkaline protease, Aspergillus oryzae triosephosphate isomerase, Aspergillus nidulans acetamidase, Fusariumvenenatum amyloglucosidase (WO 00/56900), Fusarium venenatum Daria (WO00/56900), Fusarium venenatum Quinn (WO 00/56900), Fusarium oxysporumtrypsin-like protease (WO 96/00787), Trichoderma reeseibeta-glucosidase, Trichoderma reesei cellobiohydrolase I, Trichodermareesei cellobiohydrolase II, Trichoderma reesei endoglucanase I,Trichoderma reesei endoglucanase II, Trichoderma reesei endoglucanaseIII, Trichoderma reesei endoglucanase IV, Trichoderma reeseiendoglucanase V, Trichoderma reesei xylanase I, Trichoderma reeseixylanase II, Trichoderma reesei beta-xylosidase, as well as the NA2-tpipromoter (a modified promoter including a gene encoding a neutralalpha-amylase in Aspergilli in which the untranslated leader has beenreplaced by an untranslated leader from a gene encoding triose phosphateisomerase in Aspergilli; non-limiting examples include modifiedpromoters including the gene encoding neutral alpha-amylase inAspergillus niger in which the untranslated leader has been replaced byan untranslated leader from the gene encoding triose phosphate isomerasein Aspergillus nidulans or Aspergillus oryzae); and mutant, truncated,and hybrid promoters thereof.

In a yeast host, useful promoters are obtained from the genes forSaccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiaegalactokinase (GAL1), Saccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH1,ADH2/GAP),Saccharomyces cerevisiae triose phosphate isomerase (TPI), Saccharomycescerevisiae metallothionein (CUP1), and Saccharomyces cerevisiae3-phosphoglycerate kinase. Other useful promoters for yeast host cellsare described by Romanos et al., 1992, Yeast 8: 423-488.

The control sequence may also be a suitable transcription terminatorsequence, a sequence recognized by a host cell to terminatetranscription. The terminator sequence is operably linked to the 3′terminus of the nucleotide sequence encoding the polypeptide. Anyterminator that is functional in the host cell of choice may be used inthe present invention.

Preferred terminators for filamentous fungal host cells are obtainedfrom the genes for Aspergillus oryzae TAKA amylase, Aspergillus nigerglucoamylase, Aspergillus nidulans anthranilate synthase, Aspergillusniger alpha-glucosidase, and Fusarium oxysporum trypsin-like protease.

Preferred terminators for yeast host cells are obtained from the genesfor Saccharomyces cerevisiae enolase, Saccharomyces cerevisiaecytochrome C (CYC1), and Saccharomyces cerevisiaeglyceraldehyde-3-phosphate dehydrogenase. Other useful terminators foryeast host cells are described by Romanos et al., 1992, supra.

The control sequence may also be a suitable leader sequence, anontranslated region of an mRNA that is important for translation by thehost cell. The leader sequence is operably linked to the 5′ terminus ofthe nucleotide sequence encoding the polypeptide. Any leader sequencethat is functional in the host cell of choice may be used in the presentinvention.

Preferred leaders for filamentous fungal host cells are obtained fromthe genes for Aspergillus oryzae TAKA amylase and Aspergillus nidulanstriose phosphate isomerase.

Suitable leaders for yeast host cells are obtained from the genes forSaccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae3-phosphoglycerate kinase, Saccharomyces cerevisiae alpha-factor, andSaccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP).

The control sequence may also be a polyadenylation sequence, a sequenceoperably linked to the 3′ terminus of the nucleotide sequence and, whentranscribed, is recognized by the host cell as a signal to addpolyadenosine residues to transcribed mRNA. Any polyadenylation sequencethat is functional in the host cell of choice may be used in the presentinvention.

Preferred polyadenylation sequences for filamentous fungal host cellsare obtained from the genes for Aspergillus oryzae TAKA amylase,Aspergillus niger glucoamylase, Aspergillus nidulans anthranilatesynthase, Fusarium oxysporum trypsin-like protease, and Aspergillusniger alpha-glucosidase.

Useful polyadenylation sequences for yeast host cells are described byGuo and Sherman, 1995, Molecular Cellular Biology 15: 5983-5990.

The control sequence may also be a signal peptide coding sequence thatcodes for an amino acid sequence linked to the amino terminus of apolypeptide and directs the encoded polypeptide into the cell'ssecretory pathway. The 5′ end of the coding sequence of the nucleotidesequence may inherently contain a signal peptide coding sequencenaturally linked in translation reading frame with the segment of thecoding region that encodes the secreted polypeptide. Alternatively, the5′ end of the coding sequence may contain a signal peptide codingsequence that is foreign to the coding sequence. The foreign signalpeptide coding sequence may be required where the coding sequence doesnot naturally contain a signal peptide coding sequence. Alternatively,the foreign signal peptide coding sequence may simply replace thenatural signal peptide coding sequence in order to enhance secretion ofthe polypeptide. However, any signal peptide coding sequence thatdirects the expressed polypeptide into the secretory pathway of a hostcell of choice, i.e., secreted into a culture medium, may be used in thepresent invention.

Effective signal peptide coding sequences for bacterial host cells arethe signal peptide coding sequences obtained from the genes for BacillusNCIB 11837 maltogenic amylase, Bacillus stearothermophilusalpha-amylase, Bacillus licheniformis subtilisin, Bacillus licheniformisbeta-lactamase, Bacillus stearothermophilus neutral proteases (nprT,nprS, nprM), and Bacillus subtilis prsA. Further signal peptides aredescribed by Simonen and Palva, 1993, Microbiological Reviews 57:109-137.

Effective signal peptide coding sequences for filamentous fungal hostcells are the signal peptide coding sequences obtained from the genesfor Aspergillus oryzae TAKA amylase, Aspergillus niger neutral amylase,Aspergillus niger glucoamylase, Rhizomucor miehei aspartic proteinase,Humicola insolens cellulase, Humicola insolens endoglucanase V, andHumicola lanuginosa lipase.

Useful signal peptides for yeast host cells are obtained from the genesfor Saccharomyces cerevisiae alpha-factor and Saccharomyces cerevisiaeinvertase. Other useful signal peptide coding sequences are described byRomanos et al., 1992, supra. The control sequence may also be apropeptide coding sequence that codes for an amino acid sequencepositioned at the amino terminus of a polypeptide. The resultantpolypeptide is known as a proenzyme or propolypeptide (or a zymogen insome cases). A propolypeptide is generally inactive and can be convertedto a mature active polypeptide by catalytic or autocatalytic cleavage ofthe propeptide from the propolypeptide. The propeptide coding sequencemay be obtained from the genes for Bacillus subtilis alkaline protease(aprE), Bacillus subtilis neutral protease (nprT), Saccharomycescerevisiae alpha-factor, Rhizomucor miehei aspartic proteinase, andMyceliophthora thermophila laccase (WO 95/33836).

Where both signal peptide and propeptide sequences are present at theamino terminus of a polypeptide, the propeptide sequence is positionednext to the amino terminus of a polypeptide and the signal peptidesequence is positioned next to the amino terminus of the propeptidesequence.

It may also be desirable to add regulatory sequences that allow theregulation of the expression of the polypeptide relative to the growthof the host cell. Examples of regulatory systems are those that causethe expression of the gene to be turned on or off in response to achemical or physical stimulus, including the presence of a regulatorycompound. Regulatory systems in prokaryotic systems include the lac,tac, and trp operator systems. In yeast, the ADH2 system or GAL1 systemmay be used. In filamentous fungi, the TAKA alpha-amylase promoter,Aspergillus niger glucoamylase promoter, and Aspergillus oryzaeglucoamylase promoter may be used as regulatory sequences. Otherexamples of regulatory sequences are those that allow for geneamplification. In eukaryotic systems, these regulatory sequences includethe dihydrofolate reductase gene that is amplified in the presence ofmethotrexate, and the metallothionein genes that are amplified withheavy metals. In these cases, the nucleotide sequence encoding thepolypeptide would be operably linked with the regulatory sequence.

Expression Vectors

The various nucleic acids and control sequences described herein may bejoined together to produce a recombinant expression vector comprising apolynucleotide encoding a cellulolytic protein, a polypeptide havingcellulolytic enhancing activity, a polypeptide having xylanase activity,or a polypeptide having cellobiohydrolase II activity, a promoter, andtranscriptional and translational stop signals. The expression vectorsmay include one or more (several) convenient restriction sites to allowfor insertion or substitution of the polynucleotide sequence encodingthe polypeptide at such sites. Alternatively, a polynucleotide encodingsuch a polypeptide may be expressed by inserting the polynucleotidesequence or a nucleic acid construct comprising the sequence into anappropriate vector for expression. In creating the expression vector,the coding sequence is located in the vector so that the coding sequenceis operably linked with the appropriate control sequences forexpression.

The recombinant expression vector may be any vector (e.g., a plasmid orvirus) that can be conveniently subjected to recombinant DNA proceduresand can bring about expression of the polynucleotide sequence. Thechoice of the vector will typically depend on the compatibility of thevector with the host cell into which the vector is to be introduced. Thevectors may be linear or closed circular plasmids.

The vector may be an autonomously replicating vector, i.e., a vectorthat exists as an extrachromosomal entity, the replication of which isindependent of chromosomal replication, e.g., a plasmid, anextrachromosomal element, a minichromosome, or an artificial chromosome.The vector may contain any means for assuring self-replication.Alternatively, the vector may be one that, when introduced into the hostcell, is integrated into the genome and replicated together with thechromosome(s) into which it has been integrated. Furthermore, a singlevector or plasmid or two or more vectors or plasmids that togethercontain the total DNA to be introduced into the genome of the host cell,or a transposon, may be used.

The vectors preferably contain one or more (several) selectable markersthat permit easy selection of transformed, transfected, transduced, orthe like cells. A selectable marker is a gene the product of whichprovides for biocide or viral resistance, resistance to heavy metals,prototrophy to auxotrophs, and the like.

Examples of bacterial selectable markers are the dal genes from Bacillussubtilis or Bacillus licheniformis, or markers that confer antibioticresistance such as ampicillin, kanamycin, chloramphenicol, ortetracycline resistance. Suitable markers for yeast host cells are ADE2,HIS3, LEU2, LYS2, MET3, TRP1, and URA3. Selectable markers for use in afilamentous fungal host cell include, but are not limited to, amdS(acetamidase), argB (ornithine carbamoyltransferase), bar(phosphinothricin acetyltransferase), hpt (hygromycinphosphotransferase), niaD (nitrate reductase), pyrG(orotidine-5′-phosphate decarboxylase), sC (sulfate adenyltransferase),and trpC (anthranilate synthase), as well as equivalents thereof.Preferred for use in an Aspergillus cell are the amdS and pyrG genes ofAspergillus nidulans or Aspergillus oryzae and the bar gene ofStreptomyces hygroscopicus.

The vectors preferably contain an element(s) that permits integration ofthe vector into the host cell's genome or autonomous replication of thevector in the cell independent of the genome.

For integration into the host cell genome, the vector may rely on thepolynucleotide's sequence encoding the polypeptide or any other elementof the vector for integration into the genome by homologous ornonhomologous recombination. Alternatively, the vector may containadditional nucleotide sequences for directing integration by homologousrecombination into the genome of the host cell at a precise location(s)in the chromosome(s). To increase the likelihood of integration at aprecise location, the integrational elements should preferably contain asufficient number of nucleic acids, such as 100 to 10,000 base pairs,preferably 400 to 10,000 base pairs, and most preferably 800 to 10,000base pairs, which have a high degree of identity to the correspondingtarget sequence to enhance the probability of homologous recombination.The integrational elements may be any sequence that is homologous withthe target sequence in the genome of the host cell. Furthermore, theintegrational elements may be non-encoding or encoding nucleotidesequences. On the other hand, the vector may be integrated into thegenome of the host cell by non-homologous recombination.

For autonomous replication, the vector may further comprise an origin ofreplication enabling the vector to replicate autonomously in the hostcell in question. The origin of replication may be any plasmidreplicator mediating autonomous replication that functions in a cell.The term “origin of replication” or “plasmid replicator” is definedherein as a nucleotide sequence that enables a plasmid or vector toreplicate in vivo.

Examples of bacterial origins of replication are the origins ofreplication of plasmids pBR322, pUC19, pACYC177, and pACYC184 permittingreplication in E. coli, and pUB110, pE194, pTA1060, and pAMβ1 permittingreplication in Bacillus.

Examples of origins of replication for use in a yeast host cell are the2 micron origin of replication, ARS1, ARS4, the combination of ARS1 andCEN3, and the combination of ARS4 and CEN6.

Examples of origins of replication useful in a filamentous fungal cellare AMA1 and ANS1 (Gems et al., 1991, Gene 98: 61-67; Cullen et al.,1987, Nucleic Acids Research 15: 9163-9175; WO 00/24883). Isolation ofthe AMA1 gene and construction of plasmids or vectors comprising thegene can be accomplished according to the methods disclosed in WO00/24883.

More than one copy of a polynucleotide encoding such a polypeptide maybe inserted into the host cell to increase production of thepolypeptide. An increase in the copy number of the polynucleotide can beobtained by integrating at least one additional copy of the sequenceinto the host cell genome or by including an amplifiable selectablemarker gene with the polynucleotide where cells containing amplifiedcopies of the selectable marker gene, and thereby additional copies ofthe polynucleotide, can be selected for by cultivating the cells in thepresence of the appropriate selectable agent.

The procedures used to ligate the elements described above to constructthe recombinant expression vectors are well known to one skilled in theart (see, e.g., Sambrook et al., 1989, supra).

Host Cells

Recombinant host cells comprising a polynucleotide encoding acellulolytic protein, a polypeptide having cellulolytic enhancingactivity, a polypeptide having xylanase activity, or a polypeptidehaving cellobiohydrolase II activity can be advantageously used in therecombinant production of the polypeptide. A vector comprising such apolynucleotide is introduced into a host cell so that the vector ismaintained as a chromosomal integrant or as a self-replicatingextra-chromosomal vector as described earlier. The term “host cell”encompasses any progeny of a parent cell that is not identical to theparent cell due to mutations that occur during replication. The choiceof a host cell will to a large extent depend upon the gene encoding thepolypeptide and its source.

The host cell may be a unicellular microorganism, e.g., a prokaryote, ora non-unicellular microorganism, e.g., a eukaryote.

The bacterial host cell may be any Gram positive bacterium or a Gramnegative bacterium. Gram positive bacteria include, but not limited to,Bacillus, Streptococcus, Streptomyces, Staphylococcus, Enterococcus,Lactobacillus, Lactococcus, Clostridium, Geobacillus, andOceanobacillus. Gram negative bacteria include, but not limited to, E.coli, Pseudomonas, Salmonella, Campylobacter, Helicobacter,Flavobacterium, Fusobacterium, Ilyobacter, Neisseria, and Ureaplasma.

The bacterial host cell may be any Bacillus cell. Bacillus cells usefulin the practice of the present invention include, but are not limitedto, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis,Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillusfirmus, Bacillus lautus, Bacillus lentus, Bacillus licheniformis,Bacillus megaterium, Bacillus pumilus, Bacillus stearothermophilus,Bacillus subtilis, and Bacillus thuringiensis cells.

In a preferred aspect, the bacterial host cell is a Bacillusamyloliquefaciens, Bacillus lentus, Bacillus licheniformis, Bacillusstearothermophilus or Bacillus subtilis cell. In a more preferredaspect, the bacterial host cell is a Bacillus amyloliquefaciens cell. Inanother more preferred aspect, the bacterial host cell is a Bacillusclausii cell. In another more preferred aspect, the bacterial host cellis a Bacillus licheniformis cell. In another more preferred aspect, thebacterial host cell is a Bacillus subtilis cell.

The bacterial host cell may also be any Streptococcus cell.Streptococcus cells useful in the practice of the present inventioninclude, but are not limited to, Streptococcus equisimilis,Streptococcus pyogenes, Streptococcus uberis, and Streptococcus equisubsp. Zooepidemicus cells.

In a preferred aspect, the bacterial host cell is a Streptococcusequisimilis cell. In another preferred aspect, the bacterial host cellis a Streptococcus pyogenes cell. In another preferred aspect, thebacterial host cell is a Streptococcus uberis cell. In another preferredaspect, the bacterial host cell is a Streptococcus equi subsp.Zooepidemicus cell.

The bacterial host cell may also be any Streptomyces cell. Streptomycescells useful in the practice of the present invention include, but arenot limited to, Streptomyces achromogenes, Streptomyces avermitilis,Streptomyces coelicolor, Streptomyces griseus, and Streptomyces lividanscells.

In a preferred aspect, the bacterial host cell is a Streptomycesachromogenes cell. In another preferred aspect, the bacterial host cellis a Streptomyces avermitilis cell. In another preferred aspect, thebacterial host cell is a Streptomyces coelicolor cell. In anotherpreferred aspect, the bacterial host cell is a Streptomyces griseuscell. In another preferred aspect, the bacterial host cell is aStreptomyces lividans cell.

The introduction of DNA into a Bacillus cell may, for instance, beeffected by protoplast transformation (see, e.g., Chang and Cohen, 1979,Molecular General Genetics 168: 111-115), by using competent cells (see,e.g., Young and Spizizen, 1961, Journal of Bacteriology 81: 823-829, orDubnau and Davidoff-Abelson, 1971, Journal of Molecular Biology 56:209-221), by electroporation (see, e.g., Shigekawa and Dower, 1988,Biotechniques 6: 742-751), or by conjugation (see, e.g., Koehler andThorne, 1987, Journal of Bacteriology 169: 5271-5278). The introductionof DNA into an E. coli cell may, for instance, be effected by protoplasttransformation (see, e.g., Hanahan, 1983, J. Mol. Biol. 166: 557-580) orelectroporation (see, e.g., Dower et al., 1988, Nucleic Acids Res. 16:6127-6145). The introduction of DNA into a Streptomyces cell may, forinstance, be effected by protoplast transformation and electroporation(see, e.g., Gong et al., 2004, Folia Microbiol. (Praha) 49: 399-405), byconjugation (see, e.g., Mazodier et al., 1989, J. Bacteriol. 171:3583-3585), or by transduction (see, e.g., Burke et al., 2001, Proc.Natl. Acad. Sci. USA 98: 6289-6294). The introduction of DNA into aPseudomonas cell may, for instance, be effected by electroporation (see,e.g., Choi et al., 2006, J. Microbiol. Methods 64: 391-397) or byconjugation (see, e.g., Pinedo and Smets, 2005, Appl. Environ.Microbiol. 71: 51-57). The introduction of DNA into a Streptococcus cellmay, for instance, be effected by natural competence (see, e.g., Perryand Kuramitsu, 1981, Infect. Immun. 32: 1295-1297), by protoplasttransformation (see, e.g., Catt and Jollick, 1991, Microbios. 68:189-207, by electroporation (see, e.g., Buckley et al., 1999, Appl.Environ. Microbiol. 65: 3800-3804) or by conjugation (see, e.g.,Clewell, 1981, Microbiol. Rev. 45: 409-436). However, any method knownin the art for introducing DNA into a host cell can be used.

The host cell may also be a eukaryote, such as a mammalian, insect,plant, or fungal cell.

In a preferred aspect, the host cell is a fungal cell. “Fungi” as usedherein includes the phyla Ascomycota, Basidiomycota, Chytridiomycota,and Zygomycota (as defined by Hawksworth et al., In, Ainsworth andBisby's Dictionary of The Fungi, 8th edition, 1995, CAB International,University Press, Cambridge, UK) as well as the Oomycota (as cited inHawksworth et al., 1995, supra, page 171) and all mitosporic fungi(Hawksworth et al., 1995, supra).

In a more preferred aspect, the fungal host cell is a yeast cell.“Yeast” as used herein includes ascosporogenous yeast (Endomycetales),basidiosporogenous yeast, and yeast belonging to the Fungi Imperfecti(Blastomycetes). Since the classification of yeast may change in thefuture, for the purposes of this invention, yeast shall be defined asdescribed in Biology and Activities of Yeast (Skinner, F. A., Passmore,S. M., and Davenport, R. R., eds, Soc. App. Bacteriol. Symposium SeriesNo. 9, 1980).

In an even more preferred aspect, the yeast host cell is a Candida,Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, orYarrowia cell.

In a most preferred aspect, the yeast host cell is a Saccharomycescarlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus,Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomycesnorbensis, or Saccharomyces oviformis cell. In another most preferredaspect, the yeast host cell is a Kluyveromyces lactis cell. In anothermost preferred aspect, the yeast host cell is a Yarrowia lipolyticacell.

In another more preferred aspect, the fungal host cell is a filamentousfungal cell. “Filamentous fungi” include all filamentous forms of thesubdivision Eumycota and Oomycota (as defined by Hawksworth et al.,1995, supra). The filamentous fungi are generally characterized by amycelial wall composed of chitin, cellulose, glucan, chitosan, mannan,and other complex polysaccharides. Vegetative growth is by hyphalelongation and carbon catabolism is obligately aerobic. In contrast,vegetative growth by yeasts such as Saccharomyces cerevisiae is bybudding of a unicellular thallus and carbon catabolism may befermentative.

In an even more preferred aspect, the filamentous fungal host cell is anAcremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis,Chrysosporium, Coprinus, Coriolus, Cryptococcus, Filibasidium, Fusarium,Humicola, Magnaporthe, Mucor, Myceliophthora, Neocallimastix,Neurospora, Paecilomyces, Penicillium, Phanerochaete, Phlebia,Piromyces, Pleurotus, Schizophyllum, Talaromyces, Thermoascus,Thielavia, Tolypocladium, Trametes, or Trichoderma cell.

In a most preferred aspect, the filamentous fungal host cell is anAspergillus awamori, Aspergillus fumigatus, Aspergillus foetidus,Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger orAspergillus oryzae cell. In another most preferred aspect, thefilamentous fungal host cell is a Fusarium bactridioides, Fusariumcerealis, Fusarium crookwellense, Fusarium culmorum, Fusariumgraminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi,Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusariumsambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusariumsulphureum, Fusarium torulosum, Fusarium trichothecioides, or Fusariumvenenatum cell. In another most preferred aspect, the filamentous fungalhost cell is a Bjerkandera adusta, Ceriporiopsis aneirina, Ceriporiopsisaneirina, Ceriporiopsis caregiea, Ceriporiopsis gilvescens,Ceriporiopsis pannocinta, Ceriporiopsis rivulosa, Ceriporiopsis subrufa,Ceriporiopsis subvermispora, Chrysosporium keratinophilum, Chrysosporiumlucknowense, Chrysosporium tropicum, Chrysosporium merdarium,Chrysosporium inops, Chrysosporium pannicola, Chrysosporiumqueenslandicum, Chrysosporium zonatum, Coprinus cinereus, Coriolushirsutus, Humicola insolens, Humicola lanuginosa, Mucor miehei,Myceliophthora thermophila, Neurospora crassa, Penicillium purpurogenum,Phanerochaete chrysosporium, Phlebia radiata, Pleurotus eryngii,Thielavia terrestris, Trametes villosa, Trametes versicolor, Trichodermaharzianum, Trichoderma koningii, Trichoderma longibrachiatum,Trichoderma reesei, or Trichoderma viride cell.

Fungal cells may be transformed by a process involving protoplastformation, transformation of the protoplasts, and regeneration of thecell wall in a manner known per se. Suitable procedures fortransformation of Aspergillus and Trichoderma host cells are describedin EP 238 023 and Yelton et al., 1984, Proceedings of the NationalAcademy of Sciences USA 81: 1470-1474. Suitable methods for transformingFusarium species are described by Malardier et al., 1989, Gene 78:147-156, and WO 96/00787. Yeast may be transformed using the proceduresdescribed by Becker and Guarente, In Abelson, J. N. and Simon, M. I.,editors, Guide to Yeast Genetics and Molecular Biology, Methods inEnzymology, Volume 194, pp 182-187, Academic Press, Inc., New York; Itoet al., 1983, Journal of Bacteriology 153: 163; and Hinnen et al., 1978,Proceedings of the National Academy of Sciences USA 75: 1920.

Methods of Production

Methods for producing a cellulolytic protein, a polypeptide havingcellulolytic enhancing activity, a polypeptide having xylanase activity,or a polypeptide having cellobiohydrolase II activity, comprise (a)cultivating a cell, which in its wild-type form is capable of producingthe polypeptide, under conditions conducive for production of thepolypeptide; and (b) recovering the polypeptide.

Alternatively, methods for producing cellulolytic protein, a polypeptidehaving cellulolytic enhancing activity, a polypeptide having xylanaseactivity, a polypeptide having cellobiohydrolase II activity, orcombinations thereof, comprise (a) cultivating a recombinant host cellunder conditions conducive for production of the polypeptide; and (b)recovering the polypeptide.

In the production methods, the cells are cultivated in a nutrient mediumsuitable for production of the polypeptide using methods well known inthe art. For example, the cell may be cultivated by shake flaskcultivation, and small-scale or large-scale fermentation (includingcontinuous, batch, fed-batch, or solid state fermentations) inlaboratory or industrial fermentors performed in a suitable medium andunder conditions allowing the polypeptide to be expressed and/orisolated. The cultivation takes place in a suitable nutrient mediumcomprising carbon and nitrogen sources and inorganic salts, usingprocedures known in the art. Suitable media are available fromcommercial suppliers or may be prepared according to publishedcompositions (e.g., in catalogues of the American Type CultureCollection). If the polypeptide is secreted into the nutrient medium,the polypeptide can be recovered directly from the medium. If thepolypeptide is not secreted into the medium, it can be recovered fromcell lysates.

The polypeptides are detected using the methods described herein. Theresulting broth may be used as is or the polypeptide may be recoveredusing methods known in the art. For example, the polypeptide may berecovered from the nutrient medium by conventional procedures including,but not limited to, centrifugation, filtration, extraction,spray-drying, evaporation, or precipitation.

The polypeptides may be purified by a variety of procedures known in theart including, but not limited to, chromatography (e.g., ion exchange,affinity, hydrophobic, chromatofocusing, and size exclusion),electrophoretic procedures (e.g., preparative isoelectric focusing),differential solubility (e.g., ammonium sulfate precipitation),SDS-PAGE, or extraction (see, e.g., Protein Purification, J.-C. Jansonand Lars Ryden, editors, VCH Publishers, New York, 1989) to obtainsubstantially pure polypeptides.

Methods for Processing Cellulosic Material

The compositions and methods of the present invention can be used tohydrolyze (saccharify) a cellulosic material, e.g., lignocellulose, tofermentable sugars and convert the fermentable sugars to many usefulsubstances, e.g., chemicals and fuels. The production of a desiredfermentation product from cellulosic material typically involvespretreatment, enzymatic hydrolysis (saccharification), and fermentation.

The processing of cellulosic material according to the present inventioncan be accomplished using processes conventional in the art. Moreover,the methods of the present invention can be implemented using anyconventional biomass processing apparatus configured to operate inaccordance with the invention.

Hydrolysis (saccharification) and fermentation, separate orsimultaneous, include, but are not limited to, separate hydrolysis andfermentation (SHF); simultaneous saccharification and fermentation(SSF); simultaneous saccharification and cofermentation (SSCF); hybridhydrolysis and fermentation (HHF); separate hydrolysis andco-fermentation (SHCF); hybrid hydrolysis and fermentation (HHCF); anddirect microbial conversion (DMC). SHF uses separate process steps tofirst enzymatically hydrolyze cellulosic material to fermentable sugars,e.g., glucose, cellobiose, cellotriose, and pentose sugars, and thenferment the fermentable sugars to ethanol. In SSF, the enzymatichydrolysis of cellulosic material and the fermentation of sugars toethanol are combined in one step (Philippidis, G. P., 1996, Cellulosebioconversion technology, in Handbook on Bioethanol: Production andUtilization, Wyman, C. E., ed., Taylor & Francis, Washington, D.C.,179-212). SSCF involves the cofermentation of multiple sugars (Sheehan,J., and Himmel, M., 1999, Enzymes, energy and the environment: Astrategic perspective on the U.S. Department of Energy's research anddevelopment activities for bioethanol, Biotechnol. Prog. 15: 817-827).HHF involves a separate hydrolysis step, and in addition a simultaneoussaccharification and hydrolysis step, which can be carried out in thesame reactor. The steps in an HHF process can be carried out atdifferent temperatures, i.e., high temperature enzymaticsaccharification followed by SSF at a lower temperature that thefermentation strain can tolerate. DMC combines all three processes(enzyme production, hydrolysis, and fermentation) in one or more stepswhere the same organism is used to produce the enzymes for conversion ofthe cellulosic material to fermentable sugars and to convert thefermentable sugars into a final product (Lynd, L. R., Weimer, P. J., vanZyl, W. H., and Pretorius, I. S., 2002, Microbial cellulose utilization:Fundamentals and biotechnology, Microbiol. Mol. Biol. Reviews 66:506-577). It is understood herein that any method known in the artcomprising pretreatment, enzymatic hydrolysis (saccharification),fermentation, or a combination thereof can be used in the practicing themethods of the present invention.

A conventional apparatus can include a fed-batch stirred reactor, abatch stirred reactor, a continuous flow stirred reactor withultrafiltration, and/or a continuous plug-flow column reactor (Fernandade Castilhos Corazza, Flávio Faria de Moraes, Gisella Maria Zanin andIvo Neitzel, 2003, Optimal control in fed-batch reactor for thecellobiose hydrolysis, Acta Scientiarum. Technology 25: 33-38; Gusakov,A. V., and Sinitsyn, A. P., 1985, Kinetics of the enzymatic hydrolysisof cellulose: 1. A mathematical model for a batch reactor process, Enz.Microb. Technol. 7: 346-352), an attrition reactor (Ryu, S. K., and Lee,J. M., 1983, Bioconversion of waste cellulose by using an attritionbioreactor, Biotechnol. Bioeng. 25: 53-65), or a reactor with intensivestirring induced by an electromagnetic field (Gusakov, A. V., Sinitsyn,A. P., Davydkin, I. Y., Davydkin, V. Y., Protas, O. V., 1996,Enhancement of enzymatic cellulose hydrolysis using a novel type ofbioreactor with intensive stirring induced by electromagnetic field,Appl. Biochem. Biotechnol. 56: 141-153). Additional reactor typesinclude: fluidized bed, upflow blanket, immobilized, and extruder typereactors for hydrolysis and/or fermentation.

Pretreatment.

In practicing the methods of the present invention, any pretreatmentprocess known in the art can be used to disrupt plant cell wallcomponents of cellulosic material (Chandra et al., 2007, Substratepretreatment: The key to effective enzymatic hydrolysis oflignocellulosics? Adv. Biochem. Engin./Biotechnol. 108: 67-93; Galbe andZacchi, 2007, Pretreatment of lignocellulosic materials for efficientbioethanol production, Adv. Biochem. Engin./Biotechnol. 108: 41-65;Hendriks and Zeeman, 2009, Pretreatments to enhance the digestibility oflignocellulosic biomass, Bioresource Technol. 100: 10-18; Mosier et al.,2005, Features of promising technologies for pretreatment oflignocellulosic biomass, Bioresource Technol. 96: 673-686; Taherzadehand Karimi, 2008, Pretreatment of lignocellulosic wastes to improveethanol and biogas production: A review, Int. J. of Mol. Sci. 9:1621-1651; Yang and Wyman, 2008, Pretreatment: the key to unlockinglow-cost cellulosic ethanol, Biofuels Bioproducts andBiorefining-Biofpr. 2: 26-40).

The cellulosic material can also be subjected to particle sizereduction, pre-soaking, wetting, washing, or conditioning prior topretreatment using methods known in the art.

Conventional pretreatments include, but are not limited to, steampretreatment (with or without explosion), dilute acid pretreatment, hotwater pretreatment, alkaline pretreatment, lime pretreatment, wetoxidation, wet explosion, ammonia fiber explosion, organosolvpretreatment, and biological pretreatment. Additional pretreatmentsinclude ammonia percolation, ultrasound, electroporation, microwave,supercritical CO₂, supercritical H₂O, ozone, and gamma irradiationpretreatments.

The cellulosic material can be pretreated before hydrolysis and/orfermentation. Pretreatment is preferably performed prior to thehydrolysis. Alternatively, the pretreatment can be carried outsimultaneously with enzyme hydrolysis to release fermentable sugars,such as glucose, xylose, and/or cellobiose. In most cases thepretreatment step itself results in some conversion of biomass tofermentable sugars (even in absence of enzymes).

Steam Pretreatment. In steam pretreatment, cellulosic material is heatedto disrupt the plant cell wall components, including lignin,hemicellulose, and cellulose to make the cellulose and other fractions,e.g., hemicellulose, accessible to enzymes. Cellulosic material ispassed to or through a reaction vessel where steam is injected toincrease the temperature to the required temperature and pressure and isretained therein for the desired reaction time. Steam pretreatment ispreferably done at 140-230° C., more preferably 160-200° C., and mostpreferably 170-190° C., where the optimal temperature range depends onany addition of a chemical catalyst. Residence time for the steampretreatment is preferably 1-15 minutes, more preferably 3-12 minutes,and most preferably 4-10 minutes, where the optimal residence timedepends on temperature range and any addition of a chemical catalyst.Steam pretreatment allows for relatively high solids loadings, so thatcellulosic material is generally only moist during the pretreatment. Thesteam pretreatment is often combined with an explosive discharge of thematerial after the pretreatment, which is known as steam explosion, thatis, rapid flashing to atmospheric pressure and turbulent flow of thematerial to increase the accessible surface area by fragmentation (Duffand Murray, 1996, Bioresource Technology 855: 1-33; Galbe and Zacchi,2002, Appl. Microbiol. Biotechnol. 59: 618-628; U.S. Patent ApplicationNo. 20020164730). During steam pretreatment, hemicellulose acetyl groupsare cleaved and the resulting acid autocatalyzes partial hydrolysis ofthe hemicellulose to monosaccharides and oligosaccharides. Lignin isremoved to only a limited extent.

A catalyst such as H₂SO₄ or SO₂ (typically 0.3 to 3% w/w) is often addedprior to steam pretreatment, which decreases the time and temperature,increases the recovery, and improves enzymatic hydrolysis (Ballesteroset al., 2006, Appl. Biochem. Biotechnol. 129-132: 496-508; Varga et al.,2004, Appl. Biochem. Biotechnol. 113-116: 509-523; Sassner et al., 2006,Enzyme Microb. Technol. 39: 756-762).

Chemical Pretreatment The term “chemical treatment” refers to anychemical pretreatment that promotes the separation and/or release ofcellulose, hemicellulose, and/or lignin. Examples of suitable chemicalpretreatment processes include, for example, dilute acid pretreatment,lime pretreatment, wet oxidation, ammonia fiber/freeze explosion (AFEX),ammonia percolation (APR), and organosolv pretreatments.

In dilute acid pretreatment, cellulosic material is mixed with diluteacid, typically H₂SO₄, and water to form a slurry, heated by steam tothe desired temperature, and after a residence time flashed toatmospheric pressure. The dilute acid pretreatment can be performed witha number of reactor designs, e.g., plug-flow reactors, counter-currentreactors, or continuous counter-current shrinking bed reactors (Duff andMurray, 1996, supra; Schell et al., 2004, Bioresource Technol. 91:179-188; Lee et al., 1999, Adv. Biochem. Eng. Biotechnol. 65: 93-115).

Several methods of pretreatment under alkaline conditions can also beused. These alkaline pretreatments include, but are not limited to, limepretreatment, wet oxidation, ammonia percolation (APR), and ammoniafiber/freeze explosion (AFEX).

Lime pretreatment is performed with calcium carbonate, sodium hydroxide,or ammonia at low temperatures of 85-150° C. and residence times from 1hour to several days (Wyman et al., 2005, Bioresource Technol. 96:1959-1966; Mosier et al., 2005, Bioresource Technol. 96: 673-686). WO2006/110891, WO 2006/11899, WO 2006/11900, and WO 2006/110901 disclosepretreatment methods using ammonia.

Wet oxidation is a thermal pretreatment performed typically at 180-200°C. for 5-15 minutes with addition of an oxidative agent such as hydrogenperoxide or over-pressure of oxygen (Schmidt and Thomsen, 1998,Bioresource Technol. 64: 139-151; Palonen et al., 2004, Appl. Biochem.Biotechnol. 117: 1-17; Varga et al., 2004, Biotechnol. Bioeng. 88:567-574; Martin et al., 2006, J. Chem. Technol. Biotechnol. 81:1669-1677). The pretreatment is performed at preferably 1-40% drymatter, more preferably 2-30% dry matter, and most preferably 5-20% drymatter, and often the initial pH is increased by the addition of alkalisuch as sodium carbonate.

A modification of the wet oxidation pretreatment method, known as wetexplosion (combination of wet oxidation and steam explosion), can handledry matter up to 30%. In wet explosion, the oxidizing agent isintroduced during pretreatment after a certain residence time. Thepretreatment is then ended by flashing to atmospheric pressure (WO2006/032282).

Ammonia fiber explosion (AFEX) involves treating cellulosic materialwith liquid or gaseous ammonia at moderate temperatures such as 90-100°C. and high pressure such as 17-20 bar for 5-10 minutes, where the drymatter content can be as high as 60% (Gollapalli et al., 2002, Appl.Biochem. Biotechnol. 98: 23-35; Chundawat et al., 2007, Biotechnol.Bioeng. 96: 219-231; Alizadeh et al., 2005, Appl. Biochem. Biotechnol.121: 1133-1141; Teymouri et al., 2005, Bioresource Technol. 96:2014-2018). AFEX pretreatment results in the depolymerization ofcellulose and partial hydrolysis of hemicellulose. Lignin-carbohydratecomplexes are cleaved.

Organosolv pretreatment delignifies cellulosic material by extractionusing aqueous ethanol (40-60% ethanol) at 160-200° C. for 30-60 minutes(Pan et al., 2005, Biotechnol. Bioeng. 90: 473-481; Pan et al., 2006,Biotechnol. Bioeng. 94: 851-861; Kurabi et al., 2005, Appl. Biochem.Biotechnol. 121: 219-230). Sulphuric acid is usually added as acatalyst. In organosolv pretreatment, the majority of hemicellulose isremoved.

Other examples of suitable pretreatment methods are described by Schellet al., 2003, Appl. Biochem. and Biotechnol. Vol. 105-108, p. 69-85, andMosier et al., 2005, Bioresource Technology 96: 673-686, and U.S.Published Application 2002/0164730.

In one aspect, the chemical pretreatment is preferably carried out as anacid treatment, and more preferably as a continuous dilute and/or mildacid treatment. The acid is typically sulfuric acid, but other acids canalso be used, such as acetic acid, citric acid, nitric acid, phosphoricacid, tartaric acid, succinic acid, hydrogen chloride, or mixturesthereof. Mild acid treatment is conducted in the pH range of preferably1-5, more preferably 1-4, and most preferably 1-3. In one aspect, theacid concentration is in the range from preferably 0.01 to 20 wt % acid,more preferably 0.05 to 10 wt % acid, even more preferably 0.1 to 5 wt %acid, and most preferably 0.2 to 2.0 wt % acid. The acid is contactedwith cellulosic material and held at a temperature in the range ofpreferably 160-220° C., and more preferably 165-195° C., for periodsranging from seconds to minutes to, e.g., 1 second to 60 minutes.

In another aspect, pretreatment is carried out as an ammonia fiberexplosion step (AFEX pretreatment step).

In another aspect, pretreatment takes place in an aqueous slurry. Inpreferred aspects, cellulosic material is present during pretreatment inamounts preferably between 10-80 wt %, more preferably between 20-70 wt%, and most preferably between 30-60 wt %, such as around 50 wt %. Thepretreated cellulosic material can be unwashed or washed using anymethod known in the art, e.g., washed with water.

Mechanical Pretreatment: The term “mechanical pretreatment” refers tovarious types of grinding or milling (e.g., dry milling, wet milling, orvibratory ball milling).

Physical Pretreatment: The term “physical pretreatment” refers to anypretreatment that promotes the separation and/or release of cellulose,hemicellulose, and/or lignin from cellulosic material. For example,physical pretreatment can involve irradiation (e.g., microwaveirradiation), steaming/steam explosion, hydrothermolysis, andcombinations thereof.

Physical pretreatment can involve high pressure and/or high temperature(steam explosion). In one aspect, high pressure means pressure in therange of preferably about 300 to about 600 psi, more preferably about350 to about 550 psi, and most preferably about 400 to about 500 psi,such as around 450 psi. In another aspect, high temperature meanstemperatures in the range of about 100 to about 300° C., preferablyabout 140 to about 235° C. In a preferred aspect, mechanicalpretreatment is performed in a batch-process, steam gun hydrolyzersystem that uses high pressure and high temperature as defined above,e.g., a Sunds Hydrolyzer available from Sunds Defibrator AB, Sweden.

Combined Physical and Chemical Pretreatment: Cellulosic material can bepretreated both physically and chemically. For instance, thepretreatment step can involve dilute or mild acid treatment and hightemperature and/or pressure treatment. The physical and chemicalpretreatments can be carried out sequentially or simultaneously, asdesired. A mechanical pretreatment can also be included.

Accordingly, in a preferred aspect, cellulosic material is subjected tomechanical, chemical, or physical pretreatment, or any combinationthereof, to promote the separation and/or release of cellulose,hemicellulose, and/or lignin.

Biological Pretreatment: The term “biological pretreatment” refers toany biological pretreatment that promotes the separation and/or releaseof cellulose, hemicellulose, and/or lignin from cellulosic material.Biological pretreatment techniques can involve applyinglignin-solubilizing microorganisms (see, for example, Hsu, T.-A., 1996,Pretreatment of biomass, in Handbook on Bioethanol: Production andUtilization, Wyman, C. E., ed., Taylor & Francis, Washington, D.C.,179-212; Ghosh and Singh, 1993, Physicochemical and biologicaltreatments for enzymatic/microbial conversion of cellulosic biomass,Adv. Appl. Microbiol. 39: 295-333; McMillan, J. D., 1994, Pretreatinglignocellulosic biomass: a review, in Enzymatic Conversion of Biomassfor Fuels Production, Himmel, M. E., Baker, J. O., and Overend, R. P.,eds., ACS Symposium Series 566, American Chemical Society, Washington,D.C., chapter 15; Gong, C. S., Cao, N. J., Du, J., and Tsao, G. T.,1999, Ethanol production from renewable resources, in Advances inBiochemical Engineering/Biotechnology, Scheper, T., ed., Springer-VerlagBerlin Heidelberg, Germany, 65: 207-241; Olsson and Hahn-Hagerdal, 1996,Fermentation of lignocellulosic hydrolysates for ethanol production,Enz. Microb. Tech. 18: 312-331; and Vallander and Eriksson, 1990,Production of ethanol from lignocellulosic materials: State of the art,Adv. Biochem. Eng./Biotechnol. 42: 63-95).

Saccharification.

In the hydrolysis step, also known as saccharification, the pretreatedcellulosic material is hydrolyzed to break down cellulose andalternatively also hemicellulose to fermentable sugars, such as glucose,cellobiose, xylose, xylulose, arabinose, mannose, galactose, and/orsoluble oligosaccharides. The hydrolysis is performed enzymaticallyusing an enzyme composition in the presence of a polypeptide havingcellobiohydrolase II activity and a polypeptide having xylanaseactivity. The composition can further comprise one or morehemicellulolytic enzymes. The enzymes of the compositions can also beadded sequentially.

Enzymatic hydrolysis is preferably carried out in a suitable aqueousenvironment under conditions that can be readily determined by oneskilled in the art. In a preferred aspect, hydrolysis is performed underconditions suitable for the activity of the enzyme(s), i.e., optimal forthe enzyme(s). The hydrolysis can be carried out as a fed batch orcontinuous process where the pretreated cellulosic material (substrate)is fed gradually to, for example, an enzyme containing hydrolysissolution.

The saccharification is generally performed in stirred-tank reactors orfermentors under controlled pH, temperature, and mixing conditions.Suitable process time, temperature and pH conditions can readily bedetermined by one skilled in the art. For example, the saccharificationcan last up to 200 hours, but is typically performed for preferablyabout 12 to about 96 hours, more preferably about 16 to about 72 hours,and most preferably about 24 to about 48 hours. The temperature is inthe range of preferably about 25° C. to about 70° C., more preferablyabout 30° C. to about 65° C., and more preferably about 40° C. to 60°C., in particular about 50° C. The pH is in the range of preferablyabout 3 to about 8, more preferably about 3.5 to about 7, and mostpreferably about 4 to about 6, in particular about pH 5. The dry solidscontent is in the range of preferably about 5 to about 50 wt %, morepreferably about 10 to about 40 wt %, and most preferably about 20 toabout 30 wt %.

The enzyme composition preferably comprises a polypeptide havingcellobiohydrolase II activity, a polypeptide having xylanase activity,and one or more (several) cellulolytic proteins selected from the groupconsisting of an endoglucanase, a cellobiohydrolase, and abeta-glucosidase.

The optimum amounts of the enzymes and polypeptides having cellulolyticenhancing activity depend on several factors including, but not limitedto, the mixture of component cellulolytic proteins, the cellulosicsubstrate, the concentration of cellulosic substrate, thepretreatment(s) of the cellulosic substrate, temperature, time, pH, andinclusion of fermenting organism (e.g., yeast for SimultaneousSaccharification and Fermentation).

In one aspect, an effective amount of cellulolytic protein(s) tocellulosic material is about 0.5 to about 50 mg, preferably at about 0.5to about 40 mg, more preferably at about 0.5 to about 25 mg, morepreferably at about 0.75 to about 20 mg, more preferably at about 0.75to about 15 mg, even more preferably at about 0.5 to about 10 mg, andmost preferably at about 2.5 to about 10 mg per g of cellulosicmaterial.

In another aspect, an effective amount of polypeptide(s) havingcellulolytic enhancing activity to cellulosic material is about 0.01 toabout 50.0 mg, preferably about 0.01 to about 40 mg, more preferablyabout 0.01 to about 30 mg, more preferably about 0.01 to about 20 mg,more preferably about 0.01 to about 10 mg, more preferably about 0.01 toabout 5 mg, more preferably at about 0.025 to about 1.5 mg, morepreferably at about 0.05 to about 1.25 mg, more preferably at about0.075 to about 1.25 mg, more preferably at about 0.1 to about 1.25 mg,even more preferably at about 0.15 to about 1.25 mg, and most preferablyat about 0.25 to about 1.0 mg per g of cellulosic material.

In another aspect, an effective amount of polypeptide(s) havingcellulolytic enhancing activity to cellulolytic protein(s) is about0.005 to about 1.0 g, preferably at about 0.01 to about 1.0 g, morepreferably at about 0.15 to about 0.75 g, more preferably at about 0.15to about 0.5 g, more preferably at about 0.1 to about 0.5 g, even morepreferably at about 0.1 to about 0.5 g, and most preferably at about0.05 to about 0.2 g per g of cellulolytic protein(s).

In another aspect, an effective amount of polypeptide(s) havingcellobiohydrolase II activity to cellulosic material is about 0.01 toabout 50.0 mg, preferably about 0.01 to about 40 mg, more preferablyabout 0.01 to about 30 mg, more preferably about 0.01 to about 20 mg,more preferably about 0.01 to about 10 mg, more preferably about 0.01 toabout 5 mg, more preferably at about 0.025 to about 1.5 mg, morepreferably at about 0.05 to about 1.25 mg, more preferably at about0.075 to about 1.25 mg, more preferably at about 0.1 to about 1.25 mg,even more preferably at about 0.15 to about 1.25 mg, and most preferablyat about 0.25 to about 1.0 mg per g of cellulosic material.

In another aspect, an effective amount of polypeptide(s) havingcellobiohydrolase II activity to cellulolytic protein(s) is about 0.005to about 1.0 g, preferably at about 0.01 to about 1.0 g, more preferablyat about 0.15 to about 0.75 g, more preferably at about 0.15 to about0.5 g, more preferably at about 0.1 to about 0.5 g, even more preferablyat about 0.1 to about 0.5 g, and most preferably at about 0.05 to about0.2 g per g of cellulolytic protein(s).

In another aspect, an effective amount of polypeptide(s) having xylanaseactivity to cellulosic material is about 0.01 to about 50.0 mg,preferably about 0.01 to about 40 mg, more preferably about 0.01 toabout 30 mg, more preferably about 0.01 to about 20 mg, more preferablyabout 0.01 to about 10 mg, more preferably about 0.01 to about 5 mg,more preferably at about 0.025 to about 1.5 mg, more preferably at about0.025 to about 1.25 mg, more preferably at about 0.05 to about 1.25 mg,more preferably at about 0.05 to about 1.25 mg, even more preferably atabout 0.05 to about 1.0 mg, and most preferably at about 0.05 to about0.75 mg per g of cellulosic material.

In another aspect, an effective amount of polypeptide(s) having xylanaseactivity to cellulolytic protein(s) is about 0.005 to about 1.0 g,preferably at about 0.01 to about 1.0 g, more preferably at about 0.15to about 0.75 g, more preferably at about 0.15 to about 0.5 g, morepreferably at about 0.1 to about 0.5 g, even more preferably at about0.1 to about 0.5 g, and most preferably at about 0.05 to about 0.2 g perg of cellulolytic protein(s).

Fermentation.

The fermentable sugars obtained from the pretreated and hydrolyzedcellulosic material can be fermented by one or more fermentingmicroorganisms capable of fermenting the sugars directly or indirectlyinto a desired fermentation product. “Fermentation” or “fermentationprocess” refers to any fermentation process or any process comprising afermentation step. Fermentation processes also include fermentationprocesses used in the consumable alcohol industry (e.g., beer and wine),dairy industry (e.g., fermented dairy products), leather industry, andtobacco industry. The fermentation conditions depend on the desiredfermentation product and fermenting organism and can easily bedetermined by one skilled in the art.

In the fermentation step, sugars, released from cellulosic material as aresult of the pretreatment and enzymatic hydrolysis steps, are fermentedto a product, e.g., ethanol, by a fermenting organism, such as yeast.Hydrolysis (saccharification) and fermentation can be separate orsimultaneous, as described herein.

Any suitable hydrolyzed cellulosic material can be used in thefermentation step in practicing the present invention. The material isgenerally selected based on the desired fermentation product, i.e., thesubstance to be obtained from the fermentation, and the processemployed, as is well known in the art.

The term “fermentation medium” is understood herein to refer to a mediumbefore the fermenting microorganism(s) is(are) added, such as, a mediumresulting from a saccharification process, as well as a medium used in asimultaneous saccharification and fermentation process (SSF).

“Fermenting microorganism” refers to any microorganism, includingbacterial and fungal organisms, suitable for use in a desiredfermentation process to produce a fermentation product. The fermentingorganism can be C₆ and/or C₅ fermenting organisms, or a combinationthereof. Both C₆ and C₅ fermenting organisms are well known in the art.Suitable fermenting microorganisms are able to ferment, i.e., convert,sugars, such as glucose, xylose, xylulose, arabinose, maltose, mannose,galactose, or oligosaccharides, directly or indirectly into the desiredfermentation product.

Examples of bacterial and fungal fermenting organisms producing ethanolare described by Lin et al., 2006, Appl. Microbiol. Biotechnol. 69:627-642.

Examples of fermenting microorganisms that can ferment C6 sugars includebacterial and fungal organisms, such as yeast. Preferred yeast includesstrains of the Saccharomyces spp., preferably Saccharomyces cerevisiae.

Examples of fermenting organisms that can ferment C5 sugars includebacterial and fungal organisms, such as yeast. Preferred C5 fermentingyeast include strains of Pichia, preferably Pichia stipitis, such asPichia stipitis CBS 5773; strains of Candida, preferably Candidaboidinii, Candida brassicae, Candida sheatae, Candida diddensii, Candidapseudotropicalis, or Candida utilis.

Other fermenting organisms include strains of Zymomonas, such asZymomonas mobilis; Hansenula, such as Hansenula anomala; Kluyveromyces,such as K. fragilis; Schizosaccharomyces, such as S. pombe; and E. coli,especially E. coli strains that have been genetically modified toimprove the yield of ethanol.

In a preferred aspect, the yeast is a Saccharomyces spp. In a morepreferred aspect, the yeast is Saccharomyces cerevisiae. In another morepreferred aspect, the yeast is Saccharomyces distaticus. In another morepreferred aspect, the yeast is Saccharomyces uvarum. In anotherpreferred aspect, the yeast is a Kluyveromyces. In another morepreferred aspect, the yeast is Kluyveromyces marxianus. In another morepreferred aspect, the yeast is Kluyveromyces fragilis. In anotherpreferred aspect, the yeast is a Candida. In another more preferredaspect, the yeast is Candida boidinii. In another more preferred aspect,the yeast is Candida brassicae. In another more preferred aspect, theyeast is Candida diddensii. In another more preferred aspect, the yeastis Candida pseudotropicalis. In another more preferred aspect, the yeastis Candida utilis. In another preferred aspect, the yeast is aClavispora. In another more preferred aspect, the yeast is Clavisporalusitaniae. In another more preferred aspect, the yeast is Clavisporaopuntiae. In another preferred aspect, the yeast is a Pachysolen. Inanother more preferred aspect, the yeast is Pachysolen tannophilus. Inanother preferred aspect, the yeast is a Pichia. In another morepreferred aspect, the yeast is a Pichia stipitis. In another preferredaspect, the yeast is a Bretannomyces. In another more preferred aspect,the yeast is Bretannomyces clausenii (Philippidis, G. P., 1996,Cellulose bioconversion technology, in Handbook on Bioethanol:Production and Utilization, Wyman, C. E., ed., Taylor & Francis,Washington, D.C., 179-212).

Bacteria that can efficiently ferment hexose and pentose to ethanolinclude, for example, Zymomonas mobilis and Clostridium thermocellum(Philippidis, 1996, supra).

In a preferred aspect, the bacterium is a Zymomonas. In a more preferredaspect, the bacterium is Zymomonas mobilis. In another preferred aspect,the bacterium is a Clostridium. In another more preferred aspect, thebacterium is Clostridium thermocellum.

Commercially available yeast suitable for ethanol production includes,e.g., ETHANOL RED™ yeast (available from Fermentis/Lesaffre, USA), FALI™(available from Fleischmann's Yeast, USA), SUPERSTART™ and THERMOSACC™fresh yeast (available from Ethanol Technology, WI, USA), BIOFERM™ AFTand XR (available from NABC—North American Bioproducts Corporation, GA,USA), GERT STRAND™ (available from Gert Strand AB, Sweden), and FERMIOL™(available from DSM Specialties).

In a preferred aspect, the fermenting microorganism has been geneticallymodified to provide the ability to ferment pentose sugars, such asxylose utilizing, arabinose utilizing, and xylose and arabinoseco-utilizing microorganisms.

The cloning of heterologous genes into various fermenting microorganismshas led to the construction of organisms capable of converting hexosesand pentoses to ethanol (cofermentation) (Chen and Ho, 1993, Cloning andimproving the expression of Pichia stipitis xylose reductase gene inSaccharomyces cerevisiae, Appl. Biochem. Biotechnol. 39-40: 135-147; Hoet al., 1998, Genetically engineered Saccharomyces yeast capable ofeffectively cofermenting glucose and xylose, Appl. Environ. Microbiol.64: 1852-1859; Kotter and Ciriacy, 1993, Xylose fermentation bySaccharomyces cerevisiae, Appl. Microbiol. Biotechnol. 38: 776-783;Walfridsson et al., 1995, Xylose-metabolizing Saccharomyces cerevisiaestrains overexpressing the TKL1 and TAL1 genes encoding the pentosephosphate pathway enzymes transketolase and transaldolase, Appl.Environ. Microbiol. 61: 4184-4190; Kuyper et al., 2004, Minimalmetabolic engineering of Saccharomyces cerevisiae for efficientanaerobic xylose fermentation: a proof of principle, FEMS Yeast Research4: 655-664; Beall et al., 1991, Parametric studies of ethanol productionfrom xylose and other sugars by recombinant Escherichia coli, Biotech.Bioeng. 38: 296-303; Ingram et al., 1998, Metabolic engineering ofbacteria for ethanol production, Biotechnol. Bioeng. 58: 204-214; Zhanget al., 1995, Metabolic engineering of a pentose metabolism pathway inethanologenic Zymomonas mobilis, Science 267: 240-243; Deanda et al.,1996, Development of an arabinose-fermenting Zymomonas mobilis strain bymetabolic pathway engineering, Appl. Environ. Microbiol. 62: 4465-4470;WO 2003/062430, xylose isomerase).

In a preferred aspect, the genetically modified fermenting microorganismis Saccharomyces cerevisiae. In another preferred aspect, thegenetically modified fermenting microorganism is Zymomonas mobilis. Inanother preferred aspect, the genetically modified fermentingmicroorganism is Escherichia coli. In another preferred aspect, thegenetically modified fermenting microorganism is Klebsiella oxytoca. Inanother preferred aspect, the genetically modified fermentingmicroorganism is Kluyveromyces sp.

It is well known in the art that the organisms described above can alsobe used to produce other substances, as described herein.

The fermenting microorganism is typically added to the degradedlignocellulose or hydrolysate and the fermentation is performed forabout 8 to about 96 hours, such as about 24 to about 60 hours. Thetemperature is typically between about 26° C. to about 60° C., inparticular about 32° C. or 50° C., and at about pH 3 to about pH 8, suchas around pH 4-5, 6, or 7.

In a preferred aspect, the yeast and/or another microorganism is appliedto the degraded cellulosic material and the fermentation is performedfor about 12 to about 96 hours, such as typically 24-60 hours. In apreferred aspect, the temperature is preferably between about 20° C. toabout 60° C., more preferably about 25° C. to about 50° C., and mostpreferably about 32° C. to about 50° C., in particular about 32° C. or50° C., and the pH is generally from about pH 3 to about pH 7,preferably around pH 4-7. However, some fermenting organisms, e.g.,bacteria, have higher fermentation temperature optima. Yeast or anothermicroorganism is preferably applied in amounts of approximately 10⁵ to10¹², preferably from approximately 10⁷ to 10¹⁰, especiallyapproximately 2×10⁸ viable cell count per ml of fermentation broth.Further guidance in respect of using yeast for fermentation can be foundin, e.g., “The Alcohol Textbook” (Editors K. Jacques, T. P. Lyons and D.R. Kelsall, Nottingham University Press, United Kingdom 1999), which ishereby incorporated by reference.

For ethanol production, following the fermentation the fermented slurryis distilled to extract the ethanol. The ethanol obtained according tothe methods of the invention can be used as, e.g., fuel ethanol,drinking ethanol, i.e., potable neutral spirits, or industrial ethanol.

A fermentation stimulator can be used in combination with any of theprocesses described herein to further improve the fermentation process,and in particular, the performance of the fermenting microorganism, suchas, rate enhancement and ethanol yield. A “fermentation stimulator”refers to stimulators for growth of the fermenting microorganisms, inparticular, yeast. Preferred fermentation stimulators for growth includevitamins and minerals. Examples of vitamins include multivitamins,biotin, pantothenate, nicotinic acid, meso-inositol, thiamine,pyridoxine, para-aminobenzoic acid, folic acid, riboflavin, and VitaminsA, B, C, D, and E. See, for example, Alfenore et al., Improving ethanolproduction and viability of Saccharomyces cerevisiae by a vitaminfeeding strategy during fed-batch process, Springer-Verlag (2002), whichis hereby incorporated by reference. Examples of minerals includeminerals and mineral salts that can supply nutrients comprising P, K,Mg, S, Ca, Fe, Zn, Mn, and Cu.

Fermentation Products:

A fermentation product can be any substance derived from thefermentation. The fermentation product can be, without limitation, analcohol (e.g., arabinitol, butanol, ethanol, glycerol, methanol,1,3-propanediol, sorbitol, and xylitol); an organic acid (e.g., aceticacid, acetonic acid, adipic acid, ascorbic acid, citric acid,2,5-diketo-D-gluconic acid, formic acid, fumaric acid, glucaric acid,gluconic acid, glucuronic acid, glutaric acid, 3-hydroxypropionic acid,itaconic acid, lactic acid, malic acid, malonic acid, oxalic acid,propionic acid, succinic acid, and xylonic acid); a ketone (e.g.,acetone); an amino acid (e.g., aspartic acid, glutamic acid, glycine,lysine, serine, and threonine); and a gas (e.g., methane, hydrogen (H₂),carbon dioxide (CO₂), and carbon monoxide (CO)). The fermentationproduct can also be protein as a high value product.

In a preferred aspect, the fermentation product is an alcohol. It willbe understood that the term “alcohol” encompasses a substance thatcontains one or more hydroxyl moieties. In a more preferred aspect, thealcohol is arabinitol. In another more preferred aspect, the alcohol isbutanol. In another more preferred aspect, the alcohol is ethanol. Inanother more preferred aspect, the alcohol is glycerol. In another morepreferred aspect, the alcohol is methanol. In another more preferredaspect, the alcohol is 1,3-propanediol. In another more preferredaspect, the alcohol is sorbitol. In another more preferred aspect, thealcohol is xylitol. See, for example, Gong, C. S., Cao, N. J., Du, J.,and Tsao, G. T., 1999, Ethanol production from renewable resources, inAdvances in Biochemical Engineering/Biotechnology, Scheper, T., ed.,Springer-Verlag Berlin Heidelberg, Germany, 65: 207-241; Silveira, M.M., and Jonas, R., 2002, The biotechnological production of sorbitol,Appl. Microbiol. Biotechnol. 59: 400-408; Nigam, P., and Singh, D.,1995, Processes for fermentative production of xylitol—a sugarsubstitute, Process Biochemistry 30 (2): 117-124; Ezeji, T. C., Qureshi,N. and Blaschek, H. P., 2003, Production of acetone, butanol and ethanolby Clostridium beijerinckii BA101 and in situ recovery by gas stripping,World Journal of Microbiology and Biotechnology 19 (6): 595-603.

In another preferred aspect, the fermentation product is an organicacid. In another more preferred aspect, the organic acid is acetic acid.In another more preferred aspect, the organic acid is acetonic acid. Inanother more preferred aspect, the organic acid is adipic acid. Inanother more preferred aspect, the organic acid is ascorbic acid. Inanother more preferred aspect, the organic acid is citric acid. Inanother more preferred aspect, the organic acid is 2,5-diketo-D-gluconicacid. In another more preferred aspect, the organic acid is formic acid.In another more preferred aspect, the organic acid is fumaric acid. Inanother more preferred aspect, the organic acid is glucaric acid. Inanother more preferred aspect, the organic acid is gluconic acid. Inanother more preferred aspect, the organic acid is glucuronic acid. Inanother more preferred aspect, the organic acid is glutaric acid. Inanother preferred aspect, the organic acid is 3-hydroxypropionic acid.In another more preferred aspect, the organic acid is itaconic acid. Inanother more preferred aspect, the organic acid is lactic acid. Inanother more preferred aspect, the organic acid is malic acid. Inanother more preferred aspect, the organic acid is malonic acid. Inanother more preferred aspect, the organic acid is oxalic acid. Inanother more preferred aspect, the organic acid is propionic acid. Inanother more preferred aspect, the organic acid is succinic acid. Inanother more preferred aspect, the organic acid is xylonic acid. See,for example, Chen, R., and Lee, Y. Y., 1997, Membrane-mediatedextractive fermentation for lactic acid production from cellulosicbiomass, Appl. Biochem. Biotechnol. 63-65: 435-448.

In another preferred aspect, the fermentation product is a ketone. Itwill be understood that the term “ketone” encompasses a substance thatcontains one or more ketone moieties. In another more preferred aspect,the ketone is acetone. See, for example, Qureshi and Blaschek, 2003,supra.

In another preferred aspect, the fermentation product is an amino acid.In another more preferred aspect, the organic acid is aspartic acid. Inanother more preferred aspect, the amino acid is glutamic acid. Inanother more preferred aspect, the amino acid is glycine. In anothermore preferred aspect, the amino acid is lysine. In another morepreferred aspect, the amino acid is serine. In another more preferredaspect, the amino acid is threonine. See, for example, Richard, A., andMargaritis, A., 2004, Empirical modeling of batch fermentation kineticsfor poly(glutamic acid) production and other microbial biopolymers,Biotechnology and Bioengineering 87 (4): 501-515.

In another preferred aspect, the fermentation product is a gas. Inanother more preferred aspect, the gas is methane. In another morepreferred aspect, the gas is H₂. In another more preferred aspect, thegas is CO₂. In another more preferred aspect, the gas is CO. See, forexample, Kataoka, N., A. Miya, and K. Kiriyama, 1997, Studies onhydrogen production by continuous culture system of hydrogen-producinganaerobic bacteria, Water Science and Technology 36 (6-7): 41-47; andGunaseelan V. N. in Biomass and Bioenergy, Vol. 13 (1-2), pp. 83-114,1997, Anaerobic digestion of biomass for methane production: A review.

Recovery.

The fermentation product(s) can be optionally recovered from thefermentation medium using any method known in the art including, but notlimited to, chromatography, electrophoretic procedures, differentialsolubility, distillation, or extraction. For example, alcohol isseparated from the fermented cellulosic material and purified byconventional methods of distillation. Ethanol with a purity of up toabout 96 vol. % can be obtained, which can be used as, for example, fuelethanol, drinking ethanol, i.e., potable neutral spirits, or industrialethanol.

The present invention is further described by the following examplesthat should not be construed as limiting the scope of the invention.

EXAMPLES Materials

Chemicals used as buffers and substrates were commercial products of atleast reagent grade.

Media

PDA plates were composed of 39 g of potato dextrose agar and deionizedwater to 1 liter.

Minimal medium plates were composed per liter of 6 g of NaNO₃, 0.52 g ofKCl, 1.52 g of KH₂PO₄, 1 ml of COVE trace elements solution, 20 g ofNoble agar, 20 ml of 50% glucose, 2.5 ml of MgSO₄.7H₂O, 20 ml of a 0.02%biotin solution, and deionized water to 1 liter.

COVE trace elements solution was composed of 0.04 g of Na₂B₄O₇.10H₂O,0.4 g of CuSO₄.5H₂O, 1.2 g of FeSO₄.7H₂O, 0.7 g of MnSO₄.H₂O, 0.8 g ofNa₂MoO₂.2H₂O, 10 g of ZnSO₄.7H₂O, and deionized water to 1 liter.

MDU2BP medium was composed of 45 g of maltose, 1 g of MgSO₄.7H₂O, 1 g ofNaCl, 2 g of K₂SO₄, 12 g of KH₂PO₄, 7 g of yeast extract, 2 g of urea,0.5 ml of AMG trace metals solution; pH 5.0, and deionized water to 1liter.

AMG trace metals solution was composed per liter of 14.3 g ofZnSO₄.7H₂O, 2.5 g of CuSO₄.5H₂O, 0.5 g of NiCl₂.6H₂O, 13.8 g ofFeSO₄.7H₂O, 8.5 g of MnSO₄.7H₂O, 3 g of citric acid, and deionized waterto 1 liter.

YEG medium was composed of 20 g of dextrose, 5 g of yeast extract, anddeionized water to 1 liter.

LB medium was composed of 10 g of tryptone, 5 g of yeast extract, 5 g ofsodium chloride, and deionized water to 1 liter.

YP medium was composed of 10 g of yeast extract, 20 g of Bacto peptone,and deionized water to 1 liter.

Example 1 Myceliophthora thermophila CBS 202.75 Genomic DNA Extraction

Myceliophthora thermophila CBS 202.75 was grown in 100 ml of YEG mediumin a baffled shake flask at 45° C. for 2 days with shaking at 200 rpm.Mycelia were harvested by filtration using MIRACLOTH® (Calbiochem, LaJolla, Calif., USA), washed twice in deionized water, and frozen underliquid nitrogen. Frozen mycelia were ground by mortar and pestle to afine powder, and total DNA was isolated using a DNEASY® Plant Maxi Kit(QIAGEN Inc., Valencia, Calif., USA).

Example 2 Isolation of a Full-Length Family 6 Cellobiohydrolase Gene(cel6a) from Myceliophthora thermophila CBS 202.75

A full-length Family 6 cellobiohydrolase gene (cel6a) was isolated fromMyceliophthora thermophila CBS 202.75 using a GENOMEWALKER™ UniversalKit (Clontech Laboratories, Inc., Mountain View, Calif., USA) accordingto the manufacturer's instructions. Briefly, total genomic DNA fromMyceliophthora thermophila CBS 202.75 was digested separately with fourdifferent restriction enzymes (Dra I, Eco RV, Pvu II, and Stu I) thatleave blunt ends. Each batch of digested genomic DNA was then ligatedseparately to the GENOMEWALKER™ Adaptor (Clontech Laboratories, Inc.,Mountain View, Calif., USA) to create four libraries. These librarieswere then employed as templates in PCR reactions using two gene-specificprimers shown below, one for primary PCR and one for secondary PCR. Theprimers were designed based on a partial Family 6 cellobiohydrolase gene(cel6a) sequence from Myceliophthora thermophila (WO 2004/056981).

Primer MtCel6a-R4: (SEQ ID NO: 91) 5′-ATTGGCAGCCCGGATCTGGGACAGAGTCTG-3′Pimer MtCel6a-R5: (SEQ ID NO: 92) 5′-CCGGTCATGCTAGGAATGGCGAGATTGTGG-3′

The primary amplifications were composed of 1 μl (approximately 6 ng) ofeach library as template, 0.4 mM each of dATP, dTTP, dGTP, and dCTP, 10pmol of Adaptor Primer 1 (Clontech Laboratories, Inc., Mountain View,Calif., USA), 10 pmol of primer MtCel6a-R4, 1× ADVANTAGE® GC-Melt LABuffer (Clontech Laboratories, Inc., Mountain View, Calif., USA), and1.25 units of ADVANTAGE® GC Genomic Polymerase Mix (ClontechLaboratories, Inc., Mountain View, Calif., USA) in a final volume of 25μl. The amplifications were performed using an EPPENDORF® MASTERCYCLER®5333 (Eppendorf Scientific, Inc., Westbury, N.Y., USA) programmed forpre-denaturing at 94° C. for 1 minute; 7 cycles each at a denaturingtemperature of 94° C. for 30 seconds; annealing and elongation at 72° C.for 5 minutes; and 32 cycles each at 67° C. for 5 minutes.

The secondary ampliifications were composed of 1 μl of each primary PCRproduct as template, 0.4 mM each of dATP, dTTP, dGTP, and dCTP, 10 pmolof Adaptor Primer 2 (Clontech Laboratories, Inc., Mountain View, Calif.,USA), 10 pmol of primer MtCel6a-R5, 1× ADVANTAGE® GC-Melt LA Buffer, and1.25 units of ADVANTAGE® GC Genomic Polymerase Mix in a final volume of25 μl. The amplifications were performed using an EPPENDORF®MASTERCYCLER® 5333 programmed for pre-denaturing at 94° C. for 1 minute;5 cycles each at a denaturing temperature of 94° C. for 30 seconds;annealing and elongation at 72° C. for 5 minutes; and 20 cycles at 67°C. for 5 minutes.

The reaction products were isolated by 1.0% agarose gel electrophoresisusing 40 mM Tris base-20 mM sodium acetate-1 mM disodium EDTA (TAE)buffer where a 3.5 kb product band from the Eco RV library was excisedfrom the gel, purified using a QIAQUICK® Gel Extraction Kit (QIAGEN,Valencia, Calif., USA) according to the manufacturer's instructions, andsequenced.

Example 3 Characterization of the Myceliophthora thermophila CBS 202.75Genomic Sequence Encoding a Family 6 Cellobiohydrolase II

DNA sequencing of the 3.5 kb PCR fragment was performed with aPerkin-Elmer Applied Biosystems Model 377 XL Automated DNA Sequencer(Perkin-Elmer/Applied Biosystems, Inc., Foster City, Calif., USA) usingdye-terminator chemistry (Giesecke et al., 1992, Journal of VirologyMethods 38: 47-60) and primer walking strategy. The following genespecific primers were used for sequencing:

MtCel6a-F2: (SEQ ID NO: 93) 5′-GCTGTAAACTGCGAATGGGTTCAG-3′ MtCel6a-F3:(SEQ ID NO: 94) 5′-GGGTCCCACATGCTGCGCCT-3′ MtCel6a-R8: (SEQ ID NO: 95)5′-AAAATTCACGAGACGCCGGG-3′

Nucleotide sequence data were scrutinized for quality and all sequenceswere compared to each other with assistance of PHRED/PHRAP software(University of Washington, Seattle, Wash., USA). The 3.5 kb sequence wascompared and aligned with a partial Family 6 cellobiohydrolase gene(cel6a) sequence from Myceliophthora thermophila (WO 2004/056981).

A gene model for the Myceliophthora thermophila sequence was constructedbased on similarity of the encoded protein to homologous glycosidehydrolase Family 6 proteins from Thielavia terrestris, Chaetomiumthermophilum, Humicola insolens and Trichoderma reesei. The nucleotidesequence and deduced amino acid sequence are shown in SEQ ID NO: 29 andSEQ ID NO: 30, respectively. The genomic fragment encodes a polypeptideof 482 amino acids, interrupted by 3 introns of 96, 87, and 180 bp. The% G+C content of the gene and the mature coding sequence are 61.6% and64%, respectively. Using the SignalP software program (Nielsen et al.,1997, Protein Engineering 10:1-6), a signal peptide of 17 residues waspredicted. The predicted mature protein contains 465 amino acids with amolecular mass of 49.3 kDa.

A comparative pairwise global alignment of amino acid sequences wasdetermined using the Needleman-Wunsch algorithm (Needleman and Wunsch,1970, J. Mol. Biol. 48: 443-453) as implemented in the Needle program ofEMBOSS with gap open penalty of 10, gap extension penalty of 0.5, andthe EBLOSUM62 matrix. The alignment showed that the deduced amino acidsequence of the Myceliophthora thermophila gene encoding the CEL6Amature polypeptide having cellobiohydrolase activity shared 78.6% and77.6% identity (excluding gaps) to the deduced amino acid sequences oftwo glycoside hydrolase Family 6 proteins from Chaetomium thermophilumand Humicola insolens, respectively (GeneSeqP accession numbers ADP84824and AAW44853, respectively).

Example 4 Cloning of the Myceliophthora thermophila CBS 202.75Cellobiohydrolase Gene (cel6a) and Construction of an Aspergillus oryzaeExpression Vector

Two synthetic oligonucleotide primers shown below were designed to PCRamplify the Myceliophthora thermophila cellobiohydrolase gene from thegenomic DNA prepared in Example 1. An IN-FUSION™ Cloning Kit (BDBiosciences, Palo Alto, Calif., USA) was used to clone the fragmentdirectly into the expression vector pAILo2 (WO 2004/099228), without theneed for restriction digestion and ligation.

MtCel6a-F4: (SEQ ID NO: 96) 5′-ACTGGATTTACCATGGCCAAGAAGCTTTTCATCACC-3′MtCel6a-R9: (SEQ ID NO: 97) 5′-TCACCTCTAGTTAATTAATTAGAAGGGCGGGTTGGCGT-3′Bold letters represent coding sequence. The remaining sequence ishomologous to insertion sites of pAILo2.

Fifty picomoles of each of the primers above were used in a PCR reactioncomposed of 100 ng of Myceliophthora thermophila genomic DNA, 1×ADVANTAGE® GC-Melt LA Buffer, 0.4 mM each of dATP, dTTP, dGTP, and dCTP,and 1.25 units of ADVANTAGE® GC Genomic Polymerase Mix in a final volumeof 25 μl. The amplification was performed using an EPPENDORF®MASTERCYCLER® 5333 programmed for 1 cycle at 94° C. for 1 minutes; and30 cycles each at 94° C. for 30 seconds, 62° C. for 30 seconds, and 72°C. for 2 minutes. The heat block then went to a 4° C. soak cycle.

The reaction products were isolated by 1.0% agarose gel electrophoresisusing TAE buffer where a 1842 bp product band was excised from the gel,and purified using a QIAQUICK® Gel Extraction Kit according to themanufacturer's instructions.

Plasmid pAILo2 (WO 2004/099228) was digested with Nco I and Pac I,isolated by 1.0% agarose gel electrophoresis using TAE buffer, andpurified using a QIAQUICK® Gel Extraction Kit according to themanufacturer's instructions.

The gene fragment and the digested vector were ligated together using anIN-FUSION™ Cloning Kit (BD Biosciences, Palo Alto, Calif., USA)resulting in pSMai180 in which transcription of the cellobiohydrolasegene was under the control of a hybrid of promoters from the genes forAspergillus niger neutral alpha-amylase and Aspergillus nidulans triosephosphate isomerase (NA2-tpi promoter). The ligation reaction (50 μl)was composed of 1×IN-FUSION™ Buffer (BD Biosciences, Palo Alto, Calif.,USA), 1×BSA (BD Biosciences, Palo Alto, Calif., USA), 1 μl of IN-FUSION™enzyme (diluted 1:10) (BD Biosciences, Palo Alto, Calif., USA), 100 ngof pAILo2 digested with Nco I and Pac I, and 50 ng of the Myceliophthorathermophila cel6a purified PCR product. The reaction was incubated atroom temperature for 30 minutes. One μl of the reaction was used totransform E. coli XL10 SOLOPACK® Gold Supercompetent cells (Stratagene,La Jolla, Calif., USA). An E. coli transformant containing pSMai180 wasdetected by restriction digestion and plasmid DNA was prepared using aBIOROBOT® 9600 (QIAGEN Inc., Valencia, Calif., USA). The Myceliophthorathermophila cel6a insert in pSMai180 was confirmed by DNA sequencing.

The same 1842 bp PCR fragment was cloned into pCR®2.1-TOPO® vector(Invitrogen, Carlsbad, Calif., USA) using a TOPO® TA CLONING® Kit(Invitrogen, Carlsbad, Calif., USA) to generate pSMai182. TheMyceliophthora thermophila cel6a insert in pSMai182 was confirmed by DNAsequencing. E. coli pSMai182 was deposited with the AgriculturalResearch Service Patent Culture Collection, Northern Regional ResearchCenter, Peoria, Ill., USA, on Sep. 6, 2007 and assigned accession numberNRRL B-50059.

Example 5 Expression of the Myceliophthora thermophila CBS 202.75 Family6 Cellobiohydrolase cel6a gene in Aspergillus oryzae JaL355

Aspergillus oryzae JaL355 (WO 2002/40694) protoplasts were preparedaccording to the method of Christensen et al., 1988, Bio/Technology 6:1419-1422. Three μg of pSMai180 were used to transform Aspergillusoryzae JaL355.

The transformation of Aspergillus oryzae JaL355 with pSMai180 yieldedabout 50 transformants. Twenty transformants were isolated to individualMinimal medium plates.

Confluent Minimal Medium plates of the 20 transformants were washed with5 ml of 0.01% TWEEN® 20 and inoculated separately into 25 ml of MDU2BPmedium in 125 ml glass shake flasks and incubated at 34° C. with shakingat 250 rpm. After 5 days incubation, 5 μl of supernatant from eachculture were analyzed by SDS-PAGE using a 8-16% CRITERION™ SDS-PAGE gel(Bio-Rad Laboratories, Inc. Hercules, Calif., USA) and a CRITERION® Cell(Bio-Rad Laboratories, Inc., Hercules, Calif., USA), according to themanufacturer's instructions. The resulting gel was stained withBIO-SAFE™ Coomassie Stain (Bio-Rad Laboratories, Inc., Hercules, Calif.,USA). SDS-PAGE profiles of the cultures showed that the majority of thetransformants had a major band of approximately 70 kDa.

A confluent plate of one transformant, designated transformant 14, waswashed with 10 ml of 0.01% TWEEN® 20 and inoculated into two 2 literFernbach flasks each containing 500 ml of MDU2BP medium to generatebroth for characterization of the enzyme. The culture broths wereharvested on day 5 and filtered using a 0.22 μm EXPRESS™ Plus Membrane(Millipore, Bedford, Mass., USA).

Example 6 Purification of Recombinant Myceliophthora thermophila CBS202.75 Family 6 Cellobiohydrolase II Expressed in Aspergillus oryzae

The filtered culture broth described in Example 5 was concentrated20-fold to 50 ml using an ultrafiltration device (Millipore, Bedford,Mass., USA) equipped with a 10 kDa polyethersulfone membrane at 70 psi,4° C. The concentrated broth was desalted into 20 mM Tris-HCl pH 8buffer using a HIPREP™ 26/10 desalting column (GE Healthcare,Piscataway, N.J., USA). The desalted broth was mixed with an appropriatevolume of 20 mM Tris-HCl pH 7.5 containing 3.4 M ammonium sulfate for afinal concentration of 1.2 M ammonium sulfate. The sample was loadedonto a PHENYL SUPEROSE column (HR 16/10, GE Healthcare, Piscataway,N.J., USA) equilibrated with 360 mM ammonium sulfate in 20 mM Tris-HClpH 7.5. Contaminants were eluted with a step gradient of 120 mM ammoniumsulfate followed by elution of Myceliophthora thermophila Cel6Acellobiohydrolase with 20 mM Tris-HCl pH 7.5. Fractions were analyzedusing 8-16% CRITERION™ SDS-PAGE gels and stained with GELCODE® BlueStain Reagent (Thermo Fisher Scientific, Waltham, Mass., USA).Myceliophthora thermophila Cel6A cellobiohydrolase was >90% pure asjudged by SDS-PAGE. Protein concentration was determined using a BCAProtein Assay Kit (Thermo Fisher Scientific, Waltham, Mass., USA) inwhich bovine serum albumin was used as a protein standard.

Example 7 Growth of Wild-Type Myceliophthora thermophila Strain CBS117.65

One hundred ml of shake flask medium in a 500 ml shake flask wasinoculated with two plugs from a solid plate culture of Myceliophthorathermophila strain CBS 117.65 and incubated at 45° C. on an orbitalshaker at 200 rpm for 48 hours. The shake flask medium was composed of15 g of glucose, 4 g of K₂HPO₄, 1 g of NaCl, 0.2 g of MgSO₄.7H₂O, 2 g ofMES free acid, 1 g of Bacto Peptone, 5 g of yeast extract, 2.5 g ofcitric acid, 0.2 g of CaCl₂.2H₂O, 5 g of NH₄NO₃, 1 ml of trace elementssolution, and deionized water to 1 liter. The trace elements solutionwas composed of 1.2 g of FeSO₄.7H₂O, 10 g of ZnSO₄.7H₂O, 0.7 g ofMnSO₄.H₂O, 0.4 g of CuSO₄.5H₂O, 0.4 g of Na₂B₄O₇.10H₂O, 0.8 g ofNa₂MoO₂.2H₂O, and deionized water to 1 liter. Fifty ml of the shakeflask broth was used to inoculate a 2 liter fermentation vessel.

A total of 1.8 liters of the fermentation batch medium was added to atwo liter glass jacketed fermentor (Applikon Biotechnology, Schiedam,Netherlands). The fermentation batch medium was composed per liter of 5g of yeast extract, 176 g of powdered cellulose, 2 g of glucose, 1 g ofNaCl, 1 g of Bacto Peptone, 4 g of K₂HPO₄, 0.2 g of CaCl₂.2H₂O, 0.2 g ofMgSO₄.7H₂O, 2.5 g of citric acid, 5 g of NH₄NO₃, 1.8 ml of anti-foam, 1ml of trace elements solution (above), and deionized water to 1 liter.Fermentation feed medium was composed of water and antifoam. Thefermentation feed medium was dosed at a rate of 4 g/l/hr for a period of72 hours. The fermentation vessel was maintained at a temperature of 45°C. and pH was controlled using an Applikon 1030 control system (ApplikonBiotechnology, Schiedam, Netherlands) to a set-point of 5.6+/−0.1. Airwas added to the vessel at a rate of 1 vvm and the broth was agitated byRushton impeller rotating at 1100 to 1300 rpm. At the end of thefermentation, whole broth was harvested from the vessel and centrifugedat 3000×g to remove the biomass.

Example 8 Purification of Native Cel6a Cellobiohydrolase II fromWild-Type Myceliophthora thermophila CBS 117.65 Whole Broth

The harvested broth obtained in Example 7 was centrifuged in 500 mlbottles at 13,000×g for 20 minutes at 4° C. and then sterile filteredusing a 0.22 μm polyethersulfone membrane (Millipore, Bedford, Mass.,USA). The filtered broth was concentrated and buffer exchanged with 20mM Tris-HCl pH 8.5 using a tangential flow concentrator (Pall Filtron,North Borough, Mass., USA) equipped with a 10 kDa polyethersulfonemembrane at approximately 20 psi. To decrease the amount of pigment, theconcentrate was applied to a 60 ml Q SEPHAROSE™ Big Bead column (GEHealthcare, Piscataway, N.J., USA) equilibrated with 20 mM Tris-HCl pH8.5, and step eluted with equilibration buffer containing 600 mM NaCl.Flow-through and eluate fractions were analyzed using 8-16% CRITERION™SDS-PAGE gels stained with GELCODE® Blue Stain Reagent. The flow-throughfraction contained Myceliophthora thermophila Cel6A cellobiohydrolase asjudged by the presence of a band corresponding to the apparent molecularweight of the protein by SDS-PAGE (Cel6A cellobiohydrolase:approximately 70 kDa).

The flow-through fraction was concentrated using an Amiconultrafiltration device (Millipore, Bedford, Mass., USA) equipped with a10 kDa polyethersulfone membrane at 40 psi, 4° C. and mixed with anequal volume of 20 mM Tris-HCl pH 7.5 containing 3.4 M ammonium sulfatefor a final concentration of 1.7 M ammonium sulfate. The sample wasfiltered (0.2 μM syringe filter, polyethersulfone membrane, Whatman,Maidstone, United Kingdom) to remove particulate matter prior to loadingonto a PHENYL SUPEROSE™ column (HR 16/10, GE Healthcare, Piscataway,N.J., USA) equilibrated with 1.7 M ammonium sulfate in 20 mM Tris-HCl pH7.5. Bound proteins were eluted with a 12 column volume decreasing saltgradient of 1.7 M ammonium sulfate to 0 M ammonium sulfate in 20 mMTris-HCl pH 7.5. Fractions were analyzed by 8-16% SDS-PAGE gelelectrophoresis as described above, which revealed that the Cel6Acellobiohydrolase eluted at the very end of the gradient (approximately20 mM ammonium sulfate).

Fractions containing Cel6A cellobiohydrolase II were pooled and diluted10-fold in 20 mM Tris-HCl pH 9.0 (to lower the salt and raise the pH)and then applied to a 1 ml RESOURCE™ Q column (GE Healthcare,Piscataway, N.J., USA) equilibrated with 20 mM Tris-HCl pH 9.0. Boundproteins were eluted with a 20 column volume salt gradient from 0 mM to550 mM NaCl in 20 mM Tris-HCl pH 9.0. M. thermophila Cel6Acellobiohydrolase II eluted as a single peak early in the gradient (˜25mM NaCl). The cellobiohydrolase II was >90% pure as judged by SDS-PAGE.Protein concentrations were determined using a BCA Protein Assay Kit inwhich bovine serum albumin was used as a protein standard.

Example 9 Preparation of Aspergillus aculeatus Family 10 Xylanase

Aspergillus aculeatus Family 10 xylanase (SHEARZYME® 2×; Novozymes A/S,Bagsvaerd, Denmark) was desalted into 20 mM Tris-HCl pH 8.0-150 mM NaClprior to use. Three ml of SHEARZYME® 2× was loaded onto an ECONO-PAC® 10DG desalting column (Bio-Rad Laboratories, Inc. Hercules, Calif., USA)equilibrated with 20 mM Tris-HCl pH 8.0-150 mM NaCl. Protein was elutedby the addition of 4 ml of equilibration buffer. Protein concentrationswere determined using a BCA Protein Assay Kit in which bovine serumalbumin was used as a protein standard.

Example 10 Effect of Myceliophthora thermophila CBS 117.65 Family 6Cellobiohydrolase II, Myceliophthora thermophila CBS 202.75 Family 6Cellobiohydrolase II, or Aspergillus aculeatus Family 10 Xylanase on PCSHydrolysis by a Trichoderma reesei Cellulolytic Protein Composition

Corn stover was pretreated at the U.S. Department of Energy NationalRenewable Energy Laboratory (NREL) using dilute sulfuric acid. Thefollowing conditions were used for the pretreatment: 1.4 wt. % sulfuricacid at 165° C. and 107 psi for 8 minutes. According to NREL, thewater-insoluble solids in the pretreated corn stover (PCS) contained56.5% cellulose, 4.6% hemicellulose and 28.4% lignin. Cellulose andhemicellulose were determined by a two-stage sulfuric acid hydrolysiswith subsequent analysis of sugars by high performance liquidchromatography using NREL Standard Analytical Procedure #002. Lignin wasdetermined gravimetrically after hydrolyzing the cellulose andhemicellulose fractions with sulfuric acid using NREL StandardAnalytical Procedure #003. The PCS was washed with a large volume of DDIwater on a glass filter.

Myceliophthora thermophila CBS 202.75 CEL6 cellobiohydrolase II(recombinant), Myceliophthora thermophila CBS 117.65 CEL6cellobiohydrolase II (native), or Aspergillus aculeatus xylanase wereevaluated for their ability to enhance the hydrolysis of washed PCS by aTrichoderma reesei cellulolytic protein composition (Trichoderma reeseibroth expressing Thermoascus aurantiacus GH61A polypeptide havingcellulolytic enhancing activity and Aspergillus oryzae beta-glucosidasefusion protein) obtained according to WO 2008/151079.

The hydrolysis of PCS was conducted using 2.2 ml deep-well plates(Axygen, Union City, Calif., USA) in a total reaction volume of 1.0 ml.The hydrolysis was performed with 50 mg of PCS per ml of 50 mM sodiumacetate pH 5.0 buffer containing 1 mM manganese sulfate and a fixedprotein loading of 2 mg of the Trichoderma reesei cellulolytic proteincomposition per gram of cellulose or a 20% replacement (by protein) ofthe T. reesei cellulolytic protein composition with each enzyme (3.2 mgof the Trichoderma reesei cellulolytic protein composition per g ofcellulose and 0.8 mg of each enzyme per g of cellulose). Hydrolysisassays were performed in triplicate for 72 hours at 50° C. Followinghydrolysis, samples were filtered using a 0.45 μm Multiscreen 96-wellfilter plate (Millipore, Bedford, Mass., USA) and filtrates analyzed forsugar content as described below.

When not used immediately, filtered sugary aliquots were frozen at −20°C. Sugar concentrations of samples diluted in 0.005 M H₂SO₄ weremeasured after elution by 0.005 M H₂SO₄ with 0.05% w/w benzoic acid at aflow rate of 0.6 ml per minute from a 4.6×250 mm AMINEX® HPX-87H column(Bio-Rad Laboratories, Inc. Hercules, Calif., USA) at 65° C. withquantitation by integration of the glucose and cellobiose signals byrefractive index detection (CHEMSTATION®, AGILENT® 1100 HPLC, AgilentTechnologies, Santa Clara, Calif., USA) calibrated by pure sugarsamples. The resultant equivalents were used to calculate the percentageof cellulose conversion for each reaction.

The degree of cellulose conversion was calculated using the followingequation:% conversion=[glucose concentration+1.053×(cellobioseconcentration)]/[(glucose concentration+1.053×(cellobiose concentration)in a limit digest].

The 1.053 factor for cellobiose takes into account the increase in masswhen cellobiose is converted to glucose. Sixty mg of the T. reeseicellulolytic protein preparation per g of cellulose was used for thelimit digest.

The results shown in FIG. 1 demonstrated that a 20% replacement (byprotein) of the T. reesei cellulolytic protein composition (loaded at 2mg per g of cellulose) with the M. thermophila CBS 202.75 recombinantCel6A cellobiohydrolase II or the native M. thermophila CBS 117.65 Cel6Acellobiohydrolase II improved the 72 hour hydrolysis yield by 3.1% and6.2%, respectively. Alternatively, the percent conversion with a 20%replacement of a T. reesei cellulolytic protein composition (loaded at 2mg per g of cellulose) with the M. thermophila CBS 202.75 recombinantCel6A cellobiohydrolase II was equivalent to a loading of 2.15 mg of theT. reesei cellulolytic protein composition per g of cellulose (a1.08-fold improvement). With the M. thermophila native CBS 117.65 Cel6Acellobiohydrolase II the percent conversion with a 20% replacement wasequivalent to a loading of 2.25 mg of the T. reesei cellulolytic proteincomposition per g of cellulose (a 1.13-fold improvement). A 20%replacement of the T. reesei cellulolytic protein composition (loaded at2 mg per g of cellulose) with the A. aculeatus xylanase improved thehydrolysis yield by 8.2%. The percent conversion with a 20% replacementof a T. reesei cellulolytic protein composition (loaded at 2 mg per g ofcellulose) with the A. aculeatus xylanase was equivalent to a loading of2.33 mg of the T. reesei cellulolytic protein composition per g ofcellulose (a 1.17-fold improvement).

Example 11 Effect of Myceliophthora thermophila Cel6A CellobiohydrolaseIIs and Aspergillus Aculeatus Xylanase on the Hydrolysis of PCS

Example 10 demonstrated that Myceliophthora thermophila CBS 202.75 CEL6cellobiohydrolase II (recombinant), Myceliophthora thermophila CBS117.65 CEL6 cellobiohydrolase II (native), or Aspergillus aculeatusxylanase enhanced the hydrolysis of washed PCS by the Trichoderma reeseicellulolytic protein composition (WO 2008/151079).

A PCS hydrolysis assay was performed as described in Example 10 with a20% replacement of the T. reesei cellulolytic protein composition (2 mgper g of cellulose total loading) with a 50:50 mixture of the M.thermophila CBS 202.75 CEL6 cellobiohydrolase II (recombinant) or the M.thermophila CBS 117.65 CEL6 cellobiohydrolase II (native) and the A.aculeatus xylanase (1.6 mg of the T. reesei cellulolytic proteincomposition per g cellulose; 0.2 mg of the M. thermophila CBS 202.75cellobiohydrolase II or the M. thermophila CBS 117.65 cellobiohydrolaseII per g cellulose; and 0.2 mg of the A. aculeatus xylanase per gcellulose).

As shown in FIG. 2 a mixture of Myceliophthora thermophila CBS 202.75CEL6 cellobiohydrolase II (recombinant) or Myceliophthora thermophilaCBS 117.65 CEL6 cellobiohydrolase II (native) and Aspergillus aculeatusxylanase demonstrated a 15.3% and 16.1% improvement of the 72 hourhydrolysis yield, respectively. These results corresponded to a percentconversion equivalent of 2.63 mg/g cellulose and 2.65 mg/g cellulose,respectively, of the Trichoderma reesei cellulolytic protein composition(a 1.32 and 1.33 fold improvement).

A significant enhancement in percent conversion of PCS by a cellulasemixture comprising a 10% replacement with the M. thermophila CBS 202.75CEL6 cellobiohydrolase II (recombinant) or M. thermophila CBS 117.65CEL6 cellobiohydrolase II (native) plus a 10% replacement with the A.aculeatus xylanase (M. thermophila CBS 202.75 recombinant CEL6cellobiohydrolase II+A. aculeatus xylanase: 15.3%; M. thermophila CBS117.65 native CEL6 cellobiohydrolase II+A. aculeatus xylanase: 16.1%)relative to a 20% replacement with each protein individually (M.thermophila CBS 202.75 CEL6 cellobiohydrolase II (recombinant): 3.1%; M.thermophila CBS 117.65 CEL6 cellobiohydrolase II (native): 6.2%; or A.aculeatus xylanase: 8.2%) demonstrated that the M. thermophila Cel6A(both recombinant from M. thermophila CBS 202.75 strain and native fromM. thermophila CBS 11.65 strain) and the A. aculeatus xylanase displayedsynergism in the enhancement of the T. reesei cellulolytic proteincomposition.

Example 12 Isolation of Penicillium sp

Penicillium sp. NN51602 was isolated from a compost sample of rice strawand cattle dung located in a rural village in Yunnan China on July 2007.The strain was isolated on PDA plates incubated at 45° C.

Example 13 Growth of Wild-Type Penicillium sp

One hundred ml of shake flask medium in a 500 ml shake flask wasinoculated with two plugs from a solid plate culture of Penicillium sp.NN51602 and incubated at 45° C. on an orbital shaker at 200 rpm for 48hours. The shake flask medium was composed of 15 g of glucose, 4 g ofK₂HPO₄, 1 g of NaCl, 0.2 g of MgSO₄.7H₂O, 2 g of MES free acid, 1 g ofBacto Peptone, 5 g of yeast extract, 2.5 g of citric acid, 0.2 g ofCaCl₂.2H₂O, 5 g of NH₄NO₃ 1 ml of trace elements solution, and deionizedwater to 1 liter. The trace elements solution was composed of 1.2 g ofFeSO₄.7H₂O, 10 g of ZnSO₄.7H₂O, 0.7 g of MnSO₄.H₂O, 0.4 g of CuSO₄.5H₂O,0.4 g of Na₂B₄O₇.10H₂O, 0.8 g of Na₂MoO₂.2H₂O, and deionized water to 1liter. Fifty ml of the 48 hour shake flask broth was used to inoculate a2 liter fermentation vessel.

A total of 1.8 liters of fermentation batch medium was added to a twoliter glass jacketed fermentor (Applikon Biotechnology, Schiedam,Netherlands). The fermentation batch medium was composed per liter of 5g of yeast extract, 176 g powdered cellulose, 2 g of glucose, 1 g ofNaCl, 1 g of Bacto Peptone, 4 g of K₂HPO₄, 0.2 g of CaCl₂.2H₂O, 0.2 g ofMgSO₄.7H₂O, 2.5 g of citric acid, 5 g of NH₄NO₃, 1.8 ml of anti-foam,and 1 ml of trace elements solution. Fermentation feed medium was dosedat a rate of 4 g/l/hr for a period of 72 hours. The fermentation feedmedium was composed of water and antifoam. The fermentation vessel wasmaintained at a temperature of 45° C. and pH was controlled using anApplikon 1030 control system (Applikon Biotechnology, Schiedam,Netherlands) to a set-point of 5.6+/−0.1. Air was added to the vessel ata rate of 1 vvm and the broth was agitated by a Rushton impellerrotating at 1100 to 1300 rpm. At the end of the fermentation, wholebroth was harvested from the vessel and centrifuged at 3000×g to removethe biomass.

Example 14 Purification of a Xylanase from Wild-Type Penicillium sp.Whole Broth

The harvested broth obtained in Example 13 was centrifuged in 500 mlbottles at 13,000×g for 20 minutes at 4° C. and then sterile filteredusing a 0.22 μm polyethersulfone membrane (Millipore, Bedford, Mass.,USA). The filtered broth was concentrated and buffer exchanged with 20mM Tris-HCl pH 8.5 using a tangential flow concentrator (Pall Filtron,North Borough, Mass., USA) equipped with a 10 kDa polyethersulfonemembrane at approximately 20 psi. To decrease the amount of pigment, theconcentrate was applied to a 60 ml Q SEPHAROSE™ Big Bead columnequilibrated with 20 mM Tris-HCl pH 8.5, and step eluted withequilibration buffer containing 600 mM NaCl. Flow-through and eluatefractions were examined on 8-16% CRITERION™ SDS-PAGE gels stained withGELCODE® Blue Stain Reagent. The eluate fraction contained a proteinband of approximately 50 kDa by SDS-PAGE.

The eluate fraction was concentrated using an ultrafiltration device(Millipore, Bedford, Mass., USA) equipped with a 10 kDa polyethersulfonemembrane at 40 psi, 4° C. and desalted into 20 mM Tris-HCl pH 8.5 usinga HIPREP™ 26/10 desalting column. The desalted material was loaded ontoa MONO Q™ HR 16/10 column (GE Healthcare, Piscataway, N.J., USA)equilibrated with 20 mM Tris-HCl pH 8.5. Bound proteins were eluted witha salt gradient of 0 M NaCl to 600 mM NaCl in 20 mM Tris-HCl pH 8.5 (20column volumes). Fractions were examined by SDS-PAGE as described above,which revealed that the Penicillium sp. xylanase eluted at approximately120 mM NaCl.

Fractions containing the xylanase were pooled and mixed with an equalvolume of 20 mM Tris-HCl pH 7.5 containing 3.4 M ammonium sulfate for afinal concentration of 1.7 M ammonium sulfate. The sample was filtered(0.2 μM syringe filter, polyethersulfone membrane, Whatman, Maidstone,United Kingdom) to remove particulate matter prior to loading onto aPHENYL SUPEROSE™ column (HR 16/10, GE Healthcare, Piscataway, N.J., USA)equilibrated with 1.7 M ammonium sulfate in 20 mM Tris-HCl pH 7.5. Boundproteins were eluted with a decreasing salt gradient of 1.7 M ammoniumsulfate to 0 M ammonium sulfate in 20 mM Tris-HCl pH 7.5 (15 columnvolumes). Fractions were analyzed by SDS-PAGE as described above, whichrevealed the Penicillium sp xylanase eluted at the very end of thegradient (approximately 50 mM ammonium sulfate). The Penicillium sp.xylanase was >90% pure as judged by SDS-PAGE. Protein concentrationswere determined using a BCA Protein Assay Kit in which bovine serumalbumin was used as a protein standard.

Example 15 Protein Identification of Penicillium sp. GH10B Xylanase

In-Gel Digestion of Polypeptides for Peptide Sequencing.

A MULTIPROBE® II Liquid Handling Robot (PerkinElmer Life and AnalyticalSciences, Boston, Mass., USA) was used to perform in-gel digestions. The50 kDa protein gel band (Example 14) was reduced with 50 μl of 10 mMdithiothreitol (DTT) in 100 mM ammonium bicarbonate pH 8.0 for 30minutes. Following reduction, the gel piece was alkylated with 50 μl of55 mM iodoacetamide in 100 mM ammonium bicarbonate pH 8.0 for 20minutes. The dried gel piece was allowed to swell in 25 μl of a trypsindigestion solution containing 6 ng of sequencing grade trypsin (Promega,Madison, Wis., USA) per μl of 50 mM ammonium bicarbonate pH 8 for 30minutes at room temperature, followed by an 8 hour digestion at 40° C.Each of the reaction steps described above was followed by numerouswashes and pre-washes with the appropriate solutions following themanufacturer's standard protocol. Fifty μl of acetonitrile was used tode-hydrate the gel piece between reactions and the gel piece was airdried between steps. Peptides were extracted twice with 1% formicacid/2% acetonitrile in HPLC grade water for 30 minutes. Peptideextraction solutions were transferred to a 96 well skirted PCR typeplate (ABGene, Rochester, N.Y., USA) that had been cooled to 10-15° C.and covered with a 96-well plate lid (PerkinElmer Life and AnalyticalSciences, Boston, Mass., USA) to prevent evaporation. Plates werefurther stored at 4° C. until mass spectrometry analysis could beperformed.

Protein Identification.

For de novo peptide sequencing by tandem mass spectrometry, aQ-TOFMICRO™ (Waters Micromass MS Technologies, Milford, Mass., USA), ahybrid orthogonal quadrupole time-of-flight mass spectrometer, was usedfor LC/MS/MS analysis. The Q-TOF MICRO™ is fully microprocessorcontrolled using MASSLYNX™ software version 4.1 (Waters Micromass MSTechnologies, Milford, Mass., USA). The Q-TOF MICRO™ was fitted with aNANOACQUITY UPLC® (Waters Corp, Milford, Mass., USA) for concentratingand desalting samples. Samples were loaded onto a trapping column (180μm ID×20 mm, 5 μm SYMMETRY® C18, Waters Corp, Milford, Mass., USA)fitted in the injection loop and washed with 0.1% formic acid in waterat 15 μl per minute for 1 minute using a binary solvent manager pump.Peptides were separated on a 100 μm ID×100 mm, C18, 1.7 μm, BEH130™ C18nanoflow fused capillary column (Waters Corp, Milford, Mass., USA) at aflow rate of 400 nl per minute. A step elution gradient of 1% to 85%acetonitrile in 0.1% formic acid was applied over a 30 minute interval.The column eluent was monitored at 214 nm and introduced into the Q-TOFMICRO™ through an electrospray ion source fitted with a nanosprayinterface.

Data was acquired in survey scan mode from a mass range of m/z 400 to1990 with switching criteria for MS to MS/MS to include an ion intensityof greater than 10.0 counts per second and charge states of +2, +3, and+4. Analysis spectra of up to 6 co-eluting species with a scan time of1.9 seconds and inter-scan time of 0.1 seconds could be obtained. A conevoltage of 45 volts was typically used and the collision energy wasprogrammed to vary according to the mass and charge state of the elutingpeptide and in the range of 10-60 volts. The acquired spectra werecombined, smoothed, and centered in an automated fashion and a peak listgenerated. The peak list was searched against selected databases usingPROTEINLYNX GLOBAL SERVER™ 2.3 software (Waters Micromass MSTechnologies, Milford, Mass., USA) and PEAKS Studio version 4.5 (SP1)(Bioinformatic Solutions Inc., Waterloo, Ontario, Canada). Results fromthe PROTEINLYNX GLOBAL SERVER™ and PEAKS Studio searches were evaluatedand un-identified proteins were analyzed further by evaluating the MS/MSspectrums of each ion of interest and de novo sequence was determined byidentifying the y and b ion series and matching mass differences to theappropriate amino acid.

Peptide sequences were obtained from several multiply charged ions forthe in-gel digested 50 kDa polypeptide gel band. A doubly chargedtryptic peptide ion of 403.231 m/z sequence was determined to beAla-Asn-Gly-Gln-Met(ox)-[Ile/Leu]-Arg (amino acids 97 to 103 of SEQ IDNO: 99). Another doubly charged tryptic peptide ion of 442.592 m/zsequence was determined to beAsn-His-[Ile/Leu]-Thr-Asn-Val-Val-Thr-His-Tyr-Lys (amino acids 133 to142 of SEQ ID NO: 99). Another doubly charged tryptic peptide ion of447.1993 m/z sequence was determined to be[Ile/Leu]-Val-Gln-Ser-Tyr-Gly-Ala-Arg (amino acids 215 to 222 of SEQ IDNO: 99). Another doubly charged tryptic peptide ion of 458.262 m/zsequence was determined to be Ala-Thr-Ala-Ala-Gln-Asn-[Ile/Leu]-Val-Lys(amino acids 206 to 214 of SEQ ID NO: 99). Another doubly chargedtryptic peptide ion of 663.380 m/z a partial sequence was determined tobe Ser-Gly-Gly-Asp-Gln-[Ile/Leu]-Ala-Asn-[Ile/Leu]-Ala-Lys (amino acids86 to 96 of SEQ ID NO: 99). Met(ox) is oxidized methionine. [Ile/Leu]and [Gln/Lys] cannot be distinguished because they have equivalentmasses.

Example 16 Penicillium sp. Genomic DNA Extraction

Penicillium sp. was grown on PDA plates at 37° C. to confluence. Three 4mm² squares were cut from the PDA plates, inoculated into 25 ml of YPmedium containing 2% glucose in a baffled 125 ml shake flask, andincubated at 37° C. for 2 days with shaking at 200 rpm. Mycelia wereharvested by filtration using MIRACLOTH® (Calbiochem, La Jolla, Calif.,USA), washed twice in deionized water, and frozen under liquid nitrogen.Frozen mycelia were ground, by mortar and pestle, to a fine powder, andtotal DNA was isolated using a DNEASY® Plant Maxi Kit.

Example 17 Isolation of a Partial Fragment of a Xylanase Gene fromPenicillium sp

Using the Consensus-Degenerate Hybrid Oligonucleotide Primer Program(CODEHOP; Rose et al., 1998, Nucleic Acids Research 26: 1628-1635),degenerate primers, shown below, were designed to regions of homologywith related xylanase sequences based on the identified peptidefragments described in Example 15.

Primer Penuldeg220F: (SEQ ID NO: 100) 5′-CAACGGCCAGATGYTNMGNTGYCAY-3′Protein translation for degenerate primer Penuldeg220F: NGQMXXCHPrimer Penul345R128fold: (SEQ ID NO: 101) 5′-GCGCCGTASGAYTGNACSARYTT-3′Protein translation for degenerate primer Penul345R128fold: KXVQSYG

To obtain the initial DNA fragment of the Penicillium sp. xylanase gene,gradient PCR was performed at 6 different annealing temperatures rangingfrom 45° C. to 65° C. Amplification reactions (25 μl) were composed of100 ng of Penicillium sp. genomic DNA as template, 0.4 mM each of dATP,dTTP, dGTP, and dCTP, 50 pmol each of primer Penuldeg220F and primerPenul345R128fold, 1× ADVANTAGE® GC-Melt LA Buffer, and 1.25 units ofADVANTAGE® GC Genomic Polymerase Mix. The amplifications were performedusing an EPPENDORF® MASTERCYCLER® 5333 programmed for pre-denaturing at95° C. for 1 minute; 30 cycles each at a denaturing temperature of 95°C. for 30 seconds; annealing temperature of 55° C.+/−10° C. for 30seconds (6 gradient options) and elongation at 70° C. for 1 minute; andfinal elongation at 70° C. for 5 minutes.

The reaction products were isolated by 1.0% agarose gel electrophoresisin TBE (10.8 g of Tris base, 5.5 g of boric acid and 4 ml of 0.5 M EDTApH 8.0 per liter) buffer. A PCR product band of approximately 375 bpfrom an annealing temperature of 55.8° C. was excised from the gel,purified using a QIAQUICK® Gel Extraction Kit according to themanufacturer's instructions, and sequenced with a Perkin-Elmer AppliedBiosystems Model 377 XL Automated DNA Sequencer using dye-terminatorchemistry (Giesecke et al., 1992, supra) and primer walking. A partialsequence was obtained, which encoded a peptide comprising several of thepeptide fragments identified in Example 15.

Example 18 Identification of a Full-Length Penicillium sp. Xylanase Gene

A full-length xylanase gene was identified from Penicillium sp. using aGENOMEWALKER™ Universal Kit according to the manufacturer'sinstructions. Briefly, total genomic DNA from Penicillium sp. wasdigested separately with four different restriction enzymes (Dra I, EcoRV, Pvu II, and Stu I) that leave blunt ends. Each batch of digestedgenomic DNA was then ligated separately to the GENOMEWALKER™ Adaptor tocreate four libraries. These four libraries were then employed astemplates in PCR reactions using four gene-specific primers shown below,two for a primary and secondary PCR amplifying upstream of the fragmentthrough the 5′ end encoding the N-terminus of the xylanase and two for aprimary and secondary PCR amplifying downstream of the fragment throughthe 3′ end encoding the C-terminus of the xylanase. The followingprimers were designed based on the partial xylanase gene sequence fromPenicillium sp. obtained as described in Example 17.

N-terminus: Primer PenulGSP1R (primary): (SEQ ID NO: 102)5′-GCCCTTGTAATGGGTAACGACGTTGGTGA-3′ Primer PenulGSP2R (secondary):(SEQ ID NO: 103) 5′-GCAAGCAGCGTCTCGTTGGTCCAGGATC-3′ C-terminus:Primer PenulGSP1F (primary): (SEQ ID NO: 104)5′-GGCACCTACCGCAGCAACGTCTTCTACCA-3′ Primer PenulGSP2F (secondary):(SEQ ID NO: 105) 5′-ACGGCGGCGCAGAACATCGTCAAGCT-3′

The primary amplifications were composed of 1 μl (approximately 6 ng) ofeach library as template, 0.4 mM each of dATP, dTTP, dGTP, and dCTP, 10pmol of Adaptor Primer 1, 50 pmol of primer PenulGSP1R or PenulGSP1F, 1×ADVANTAGE® GC-Melt LA Buffer, and 1.25 units of ADVANTAGE® GC GenomicPolymerase Mix in a final volume of 25 μl. The amplifications wereperformed using an EPPENDORF® MASTERCYCLER® 5333 programmed forpre-denaturing at 95° C. for 1 minute; 7 cycles each at a denaturingtemperature of 95° C. for 25 seconds; annealing and elongation at 72° C.for 5 minutes; 32 cycles each at a denaturing temperature of 95° C. for25 seconds; annealing and elongation at 67° C. for 5 minutes; and finalelongation at 67° C. for 7 minutes.

The secondary amplifications were composed of 1 μl of each primary PCRproduct as template, 0.4 mM each of dATP, dTTP, dGTP, and dCTP, 10 pmolof Adaptor Primer 2, 50 pmol of primer PenulGSP2R or PenulGSP2F, 1×ADVANTAGE® GC-Melt LA Buffer, and 1.25 units of ADVANTAGE® GC GenomicPolymerase Mix in a final volume of 25 μl. The amplifications wereperformed using an EPPENDORF® MASTERCYCLER® 5333 programmed forpre-denaturing at 95° C. for 1 minute; 5 cycles each at a denaturingtemperature of 95° C. for 25 seconds; annealing and elongation at 72° C.for 5 minutes; 20 cycles each at a denaturing temperature of 95° C. for25 seconds; annealing and elongation at 67° C. for 5 minutes; and finalelongation at 67° C. for 7 minutes.

The reaction products were isolated by 1.0% agarose gel electrophoresisusing TBE buffer. From the 5′ end PCR amplification, 4 product bandswere analyzed: a 450 bp product band from the Dra I library, a 1.6 kbproduct band from the Eco RV library, a 1.7 kb product band from the PvuII library, and a 550 bp band from the Stu I library. The 4 productbands were excised from the gel, purified using a QIAQUICK® GelExtraction Kit according to the manufacturer's instructions, andsequenced. From the 3′ end PCR amplification, 3 product bands wereanalyzed: a 450 bp product band from the Dra I library, and 600 bp and800 bp product bands from the Eco RV library. The 3 product bands wereexcised from the gel, purified using a QIAQUICK® Gel Extraction Kitaccording to the manufacturer's instructions, and sequenced.

DNA sequencing of the PCR fragments was performed with a Perkin-ElmerApplied Biosystems Model 377 XL Automated DNA Sequencer usingdye-terminator chemistry (Giesecke et al., 1992, supra) and primerwalking strategy using Adaptor Primer 2, primer PenulGSP2R, and primerPenulGSP2F for sequencing.

Nucleotide sequence data were scrutinized for quality and all sequenceswere compared to each other with assistance of PHRED/PHRAP software(University of Washington, Seattle, Wash., USA). The PCR fragmentsequence results were compared and aligned with the partial xylanasegene sequence from Penicillium sp. obtained as described in Example 17.A gene model was constructed based on the gene fragments obtained hereand in Example 17 allowing determination of the 5′ and 3′ ends of thegene with other homologous xylanases.

Example 19 Cloning of the Penicillium sp. Xylanase Gene and Constructionof an Aspergillus Niger Expression Vector

Two synthetic oligonucleotide primers shown below were designed to PCRamplify the Penicillium sp. xylanase gene from the genomic DNA preparedin Example 16. An IN-FUSION™ Cloning Kit was used to clone the fragmentdirectly into the expression vector pBM120a (WO 2006/078256).

PenulxylNCO1F: (SEQ ID NO: 106)5′-ACACAACTGGCCATGGTTCGCCTCAGTCCAGTCCTGC-3′ PenulxylPACIR:(SEQ ID NO: 107) 5′-CAGTCACCTCTAGTTATTACAGACACTGCGAGTAATACTCG-3′

Bold letters represent coding sequence. The remaining sequence ishomologous to the insertion sites of pBM120a.

Fifty picomoles of each of the primers above were used in a PCR reactioncomposed of 105 ng of Penicillium sp. genomic DNA, 1× EXPAND® Buffer 2(Roche Diagnostics Corporation, Indianapolis, Ind., USA), 0.4 mM each ofdATP, dTTP, dGTP, and dCTP, and 1 unit of EXPAND® DNA Polymerase (RocheDiagnostics Corporation, Indianapolis, Ind., USA) in a final volume of50 μl. The amplification was performed using an EPPENDORF® MASTERCYCLER®5333 programmed for 1 cycle at 95° C. for 1 minute; 30 cycles each at95° C. for 30 seconds, 63.5° C. for 30 seconds, and 72° C. for 90seconds; and a final elongation at 72° C. for 7 minutes. The heat blockthen went to a 4° C. soak cycle.

The reaction products were isolated by 1.0% agarose gel electrophoresisin TBE buffer where an approximately 1.4 kb product band was excisedfrom the gel, and purified using a QIAQUICK® Gel Extraction Kitaccording to the manufacturer's instructions.

Plasmid pBM120a was digested with Nco I and Pac I, isolated by 1.0%agarose gel electrophoresis in TBE buffer, and purified using aQIAQUICK® Gel Extraction Kit according to the manufacturer'sinstructions.

The gene fragment and the digested vector were ligated together using anIN-FUSION™ Cloning Kit resulting in pMMar31 in which transcription ofthe xylanase gene was under the control of a hybrid of promoters fromthe genes for Aspergillus niger neutral alpha-amylase and Aspergillusoryzae triose phosphate isomerase (NA2-tpi promoter). The ligationreaction (20 μl) was composed of 1×IN-FUSION™ Buffer, 1×BSA (BDBiosciences, Palo Alto, Calif., USA), 1 μl of IN-FUSION™ enzyme (diluted1:10), 132 ng of pBM120a digested with Nco I and Pac I, and 104 ng ofthe purified Penicillium sp. PCR product. The reaction was incubated atroom temperature for 30 minutes. Two μl of the reaction were used totransform E. coli XL10 SOLOPACK® Gold Ultracompetent cells (Stratagene,La Jolla, Calif., USA) according to the manufacturer's instructions.Transformants were picked into LB medium supplemented with 100 μg ofampicillin per ml and grown overnight at 37° C. Plasmid DNA was preparedfrom each of the cultures using a BIOROBOT® 9600 and submitted to DNAsequencing with a Perkin-Elmer Applied Biosystems Model 377 XL AutomatedDNA Sequencer using dye-terminator chemistry (Giesecke et al., 1992,supra) and primer walking strategy using the primers below forsequencing. One E. coli transformant was identified containing thePenicillium sp. xylanase gene. The plasmid containing the xylanase genewas designated pMMar31.

996271 Na2tpi promoter fwd: (SEQ ID NO: 108)5′-ACTCAATTTACCTCTATCCACACTT-3′ 996270 AMG rev: (SEQ ID NO: 109)5′-CTATAGCGAAATGGATTGATTGTCT-3′ Penulxyl367F: (SEQ ID NO: 110)5′-ATGTTGAGGTGCCATAATC-3′ Penulxyl1025R: (SEQ ID NO: 111)5′-TCTGGTAGTCGGTCGCCTG-3′

The same 1.4 kb PCR fragment was cloned into pCR®2.1-TOPO® using a TOPO®TA CLONING Kit to generate pMMar26. The Penicillium sp. xylanase insertin pMMar26 was confirmed by DNA sequencing. E. coli pMMar26 wasdeposited with the Agricultural Research Service Patent CultureCollection, Northern Regional Research Center, Peoria, Ill., USA, onMar. 13, 2009, and assigned accession number NRRL B-50266.

Example 20 Characterization of the Penicillium sp. Genomic SequenceEncoding a Family GH10 Xylanase

Nucleotide sequence data were scrutinized for quality and all sequenceswere compared to each other with assistance of PHRED/PHRAP software(University of Washington, Seattle, Wash., USA).

The nucleotide sequence and deduced amino acid sequence are shown in SEQID NO: 98 and SEQ ID NO: 99, respectively. The genomic fragment encodesa polypeptide of 403 amino acids, interrupted by 3 predicted introns of65, 55, and 52 base pairs. The % G+C content of the full-length codingsequence and the mature coding sequence are 60.2% and 60.0%,respectively. Using the SignalP software program (Nielsen et al., 1997,supra), a signal peptide of 23 residues was predicted. The predictedmature protein contains 380 amino acids with a predicted molecular massof 41.1 kDa. Amino acids 25 to 340 are indicative of a Family 10glycosyl hydrolase. Based on the deduced amino acid sequence, thexylanase appears to fall into the xylanase Family GH10 according toCoutinho and Henrissat, 1999, supra.

A comparative pairwise global alignment of amino acid sequences wasdetermined using the Needleman-Wunsch algorithm (Needleman and Wunsch,1970, supra) as implemented in the Needle program of EMBOSS with gapopen penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62matrix. The alignment showed that the deduced amino acid sequence of themature polypeptide of the Penicillium sp. Family GH10 xylanase geneshared 93% identity (excluding gaps) to the deduced amino acid sequenceof a Talaromyces emersonii xylanase gene (GeneSeq accession numberAAB84358).

Example 21 Effect of Myceliophthora thermophila CBS 117.65 Family 6Cellobiohydrolase II, Myceliophthora thermophila CBS 202.75 Family 6Cellobiohydrolase II, or Penicillium sp. Family 10 Xylanase on PCSHydrolysis

Myceliophthora thermophila CBS 202.75 CEL6 cellobiohydrolase II(recombinant), Myceliophthora thermophila CBS 117.65 CEL6cellobiohydrolase II (native), or Penicillium sp. Family 10 xylanasewere evaluated for their ability to enhance the hydrolysis of washed PCSby a Trichoderma reesei cellulolytic protein composition (Trichodermareesei broth expressing Thermoascus aurantiacus GH61A polypeptide havingcellulolytic enhancing activity and Aspergillus oryzae beta-glucosidasefusion protein) obtained according to WO 2008/151079.

The hydrolysis of PCS was conducted using 2.2 ml deep-well plates in atotal reaction volume of 1.0 ml. The hydrolysis was performed with 50 mgof PCS per ml of 50 mM sodium acetate pH 5.0 buffer containing 1 mMmanganese sulfate and a fixed protein loading of 2 mg of the T. reeseicellulolytic protein composition per gram of cellulose or a 20%replacement (by protein) of the T. reesei cellulolytic proteincomposition with each enzyme (1.6 mg of the T. reesei cellulolyticprotein composition per g of cellulose and 0.4 mg of each enzyme per gof cellulose). Hydrolysis assays were performed in triplicate for 72hours at 50° C. Following hydrolysis, samples were filtered with a 0.45μm Multiscreen 96-well filter plate and filtrates analyzed for sugarcontent according to Example 10.

The results shown in FIG. 3 demonstrated that a 20% replacement (byprotein) of the T. reesei cellulolytic protein composition (loaded at 2mg per g of cellulose) with the M. thermophila CBS 202.75 recombinantCel6A cellobiohydrolase II or native M. thermophila CBS 117.65 Cel6Acellobiohydrolase II improved the 72 hour hydrolysis yield by 3.1% and6.2%, respectively. Alternatively, the percent conversion with a 20%replacement of the T. reesei cellulolytic protein composition (loaded at2 mg per g of cellulose) with the M. thermophila CBS 202.75 recombinantCel6A cellobiohydrolase II was equivalent to a loading of 2.15 mg of theT. reesei cellulolytic protein composition per g of cellulose (a1.08-fold improvement). With the M. thermophila native CBS 117.65 Cel6Acellobiohydrolase II the percent conversion with a 20% replacement wasequivalent to a loading of 2.25 mg of the T. reesei cellulolytic proteincomposition per g of cellulose (a 1.13-fold improvement). A 20%replacement of the T. reesei cellulolytic protein composition (loaded at2 mg per g of cellulose) with the Penicillium sp. Family 10 xylanaseimproved the hydrolysis yield by 7.7%. The percent conversion with a 20%replacement of the T. reesei cellulolytic protein composition (loaded at2 mg per g of cellulose) with the Penicillium sp. xylanase wasequivalent to a loading of 2.32 mg of the T. reesei cellulolytic proteincomposition per g of cellulose (a 1.16-fold improvement).

Example 22 Effect of Myceliophthora thermophila CBS 202.75 Cel6ACellobiohydrolase II or Myceliophthora thermophila CBS 117.65 Cel6Acellobiohydrolase II and Penicillium sp. Family 10 Xylanase on theHydrolysis of PCS by a Trichoderma reesei Cellulase Mixture

A PCS hydrolysis assay was performed as described in Example 21 with a20% replacement of the T. reesei cellulolytic protein compositiondescribed in Example 21 (2 mg per g of cellulose total loading) with a50:50 mixture of the M. thermophila CBS 202.75 CEL6 cellobiohydrolase II(recombinant) or the M. thermophila CBS 117.65 CEL6 cellobiohydrolase II(native) and the Penicillium sp. Family 10 xylanase (1.6 mg of the T.reesei cellulolytic protein composition per g cellulose; 0.2 mg of theM. thermophila CBS 202.75 cellobiohydrolase II or the M. thermophila CBS117.65 cellobiohydrolase II per g cellulose; and 0.2 mg of thePenicillium sp. xylanase per g cellulose).

As shown in FIG. 4 a mixture of Myceliophthora thermophila CBS 202.75CEL6 cellobiohydrolase II (recombinant) or Myceliophthora thermophilaCBS 117.65 CEL6 cellobiohydrolase II (native) and Penicillium sp.xylanase demonstrated a 19.2% and 16.6% improvement of the 72 hourhydrolysis yield, respectively. These results corresponded to a percentconversion equivalent of 2.78 mg/g cellulose and 2.68 mg/g cellulose,respectively, of the Trichoderma reesei cellulolytic protein composition(a 1.39 and 1.34 fold improvement, respectively).

A significant enhancement in percent conversion of PCS by theTrichoderma reesei cellulolytic protein composition comprising a 10%replacement with the M. thermophila CBS 202.75 CEL6 cellobiohydrolase II(recombinant) or M. thermophila CBS 117.65 CEL6 cellobiohydrolase II(native) plus a 10% replacement with the Penicillium sp. xylanase (M.thermophila CBS 202.75 recombinant CEL6 cellobiohydrolase II+Penicilliumsp. xylanase: 19.2%; M. thermophila CBS 117.65 native CEL6cellobiohydrolase II+Penicillium sp. xylanase: 16.6%) relative to a 20%replacement with each protein individually (M. thermophila CBS 202.75CEL6 cellobiohydrolase II (recombinant): 3.1%; M. thermophila CBS 117.65CEL6 cellobiohydrolase II (native): 6.2%; Penicillium sp. xylanase:8.2%), demonstrated that the M. thermophila Cel6A cellobiohydrolase II(both recombinant from M. thermophila CBS 202.75 strain and native fromM. thermophila CBS 11.65 strain) and Penicillium sp. xylanase displayedsynergism in the enhancement of the T. reesei cellulolytic proteincomposition.

Example 23 Preparation of Trichoderma reesei RutC30 Cel6Acellobiohydrolase II

The Trichoderma reesei RutC30 Cel6A cellobiohydrolase II gene (SEQ IDNO: 25 [DNA sequence] and SEQ ID NO: 26 [deduced amino acid sequence])was isolated from Trichoderma reesei RutC30 as described in WO2005/056772.

The Trichoderma reesei Cel6A cellobiohydrolase II gene was expressed inFusarium venenatum using pEJG61 as an expression vector according to theprocedures described in U.S. Published Application No. 20060156437.Fermentation was performed as described in U.S. Published ApplicationNo. 20060156437.

Filtered broth was desalted and buffer-exchanged into 20 mM sodiumacetate-150 mM NaCl pH 5.0 using a HIPREP® 26/10 Desalting Columnaccording to the manufacturer's instructions. Protein concentration wasdetermined using a Microplate BCA™ Protein Assay Kit in which bovineserum albumin was used as a protein standard.

Example 24 Preparation of Thielavia terrestris NRRL 8126 Cel6ACellobiohydrolase II (CBHII)

Thielavia terrestris NRRL 8126 Cel6A cellobiohydrolase II (SEQ ID NO: 33[DNA sequence] and SEQ ID NO: 34 [deduced amino acid sequence]) wasrecombinantly prepared according to WO 2006/074435 using Trichodermareesei as a host.

Culture filtrate was desalted and buffer exchanged in 20 mM Tris-150 mMsodium chloride pH 8.5 using an ECONO-PAC® 10-DG desalting columnaccording to the manufacturer's instructions. Protein concentration wasdetermined using a Microplate BCA™ Protein Assay Kit in which bovineserum albumin was used as a protein standard.

Example 25 Effect of Trichoderma reesei Cel6A Cellobiohydrolase II orThielavia terrestris Cel6a Cellobiohydrolase II and Aspergillusaculeatus Xylanase on the Hydrolysis of PCS by a Trichoderma reeseiCellulase Mixture

To test synergy between other Cel6A cellobiohydrolase II proteins andAspergillus aculeatus xylanase, a PCS hydrolysis assay was performed (asdescribed in Example 10) with a 10% addition to the T. reeseicellulolytic protein composition (2 mg per g of cellulose total loading)of either T. reesei CEL6 cellobiohydrolase II or T. terrestris CEL6cellobiohydrolase II alone, or in combination with the A. aculeatusxylanase (2 mg of the T. reesei cellulolytic protein composition per gcellulose, 0.2 mg of the T. reesei CEL6 cellobiohydrolase II or T.terrestris CEL6 cellobiohydrolase II per g cellulose; or 2 mg of the T.reesei cellulolytic protein composition per g cellulose, 0.2 mg of theT. reesei CEL6 cellobiohydrolase II, 0.2 mg of the A. aculeatus xylanaseper g cellulose; or 2 mg of the T. reesei cellulolytic proteincomposition per g cellulose, 0.2 mg of the T. terrestris CEL6cellobiohydrolase II per g cellulose, 0.2 mg of the A. aculeatusxylanase per g cellulose).

As shown in FIG. 5, addition of T. reesei CEL6 cellobiohydrolase II, T.terrestris CEL6 cellobiohydrolase II, or A. aculeatus xylanasedemonstrated a 1.2%, 2.5%, or 11.0% improvement of the 72 hourhydrolysis yield, respectively. Addition of T. reesei CEL6cellobiohydrolase II and A. aculeatus xylanase or T. terrestris CEL6cellobiohydrolase II and A. aculeatus xylanase resulted in a 16% and 18%improvement of the 72 hour hydrolysis yield, respectively. The additionof both T. reesei CEL6 cellobiohydrolase II and A. aculeatus xylanase orT. terrestris CEL6 cellobiohydrolase II and A. aculeatus xylanaseresulted in a greater enhancement to conversion than would be expectedif the enhancements were additive [T. reesei CEL6 cellobiohydrolase IIand A. aculeatus xylanase 16% vs. 12.2% (1.2%+11.0%); T. terrestris CEL6cellobiohydrolase II and A. aculeatus xylanase 18% vs 13.5%(2.5%+11.0%)], indicating a synergistic enhancement to the PCShydrolysis activity of the T. reesei cellulolytic protein composition.

Deposit of Biological Material

The following biological materials have been deposited under the termsof the Budapest Treaty with the Agricultural Research Service PatentCulture Collection (NRRL), Northern Regional Research Center, 1815University Street, Peoria, Ill., 61604, USA, and given the followingaccession numbers:

Deposit Accession Number Date of Deposit E. coli pSMai182 NRRL B-50059Sep. 6, 2007 E. coli (pMMar26) NRRL B-50266 Mar. 13, 2009

The strains have been deposited under conditions that assure that accessto the cultures will be available during the pendency of this patentapplication to one determined by foreign patent laws to be entitledthereto. The deposits represent substantially pure cultures of thedeposited strains. The deposits are vailable as required by foreignpatent laws in countries wherein counterparts of the subjectapplication, or its progeny are filed. However, it should be understoodthat the availability of a deposit does not constitute a license topractice the subject invention in derogation of patent rights granted bygovernmental action.

The invention described and claimed herein is not to be limited in scopeby the specific aspects herein disclosed, since these aspects areintended as illustrations of several aspects of the invention. Anyequivalent aspects are intended to be within the scope of thisinvention. Indeed, various modifications of the invention in addition tothose shown and described herein will become apparent to those skilledin the art from the foregoing description. Such modifications are alsointended to fall within the scope of the appended claims. In the case ofconflict, the present disclosure including definitions will control.

What is claimed is:
 1. A method for degrading a cellulosic material,comprising: treating the cellulosic material with an enzyme compositioncomprising a CEL6 polypeptide having cellobiohydrolase II activity, aGH10 polypeptide having xylanase activity, and one or more cellulolyticenzymes selected from the group consisting of an endoglucanase, acellobiohydrolase, and a beta-glucosidase, wherein one or both of theCEL6 polypeptide having cellobiohydrolase II activity and the GH10polypeptide having xylanase activity are foreign to the one or morecellulolytic enzymes, wherein the CEL6 polypeptide havingcellobiohydrolase II activity is selected from the group consisting of:(a) a polypeptide having cellobiohydrolase II activity comprising anamino acid sequence having a sequence identity of at least 90% to themature polypeptide of SEQ ID NO: 30; (b) a polypeptide havingcellobiohydrolase II activity encoded by a polynucleotide thathybridizes under very high stringency conditions with the maturepolypeptide coding sequence of SEQ ID NO: 29 or the full-lengthcomplement thereof, wherein the very high stringency conditions aredefined as prehybridization and hybridization at 42° C. in 5×SSPE, 0.3%SDS, 200 μg/ml sheared and denatured salmon sperm DNA, and 50% formamidefor 12 to 24 hours and washing three times each for 15 minutes using2×SSC, 0.2% SDS at 70° C.; (c) a polypeptide having cellobiohydrolase IIactivity encoded by a polynucleotide comprising a nucleotide sequencehaving a sequence identity to the mature polypeptide coding sequence ofSEQ ID NO: 29 of at least 90%; and (d) a polypeptide havingcellobiohydrolase II activity comprising the mature polypeptide of SEQID NO: 30; and wherein the GH10 polypeptide having xylanase activity isselected from the group consisting of: (a) a polypeptide having xylanaseactivity comprising an amino acid sequence having a sequence identity ofat least 90% to the mature polypeptide of SEQ ID NO: 70; (b) apolypeptide having xylanase activity encoded by a polynucleotide thathybridizes under very high stringency conditions with the maturepolypeptide coding sequence of SEQ ID NO: 69 or the full-lengthcomplement thereof, wherein the very high stringency conditions aredefined as prehybridization and hybridization at 42° C. in 5×SSPE, 0.3%SDS, 200 μg/ml sheared and denatured salmon sperm DNA, and 50% formamidefor 12 to 24 hours and washing three times each for 15 minutes using2×SSC, 0.2% SDS at 70° C.; (c) a polypeptide having xylanase activityencoded by a polynucleotide comprising a nucleotide sequence having asequence identity of at least 90% to the mature polypeptide codingsequence of SEQ ID NO: 69; and (d) a polypeptide having xylanaseactivity comprising the mature polypeptide of SEQ ID NO:
 70. 2. Themethod of claim 1, wherein the one or more cellulolytic enzymes comprisea beta-glucosidase, a Trichoderma reesei cellobiohydrolase I (CEL7A), aTrichoderma reesei cellobiohydrolase II (CEL6A), and a Trichodermareesei endoglucanase I (CEL7B).
 3. The method of claim 1, wherein theone or more cellulolytic enzymes further comprise one or more enzymesselected from the group consisting of a Trichoderma reesei endoglucanaseII (CEL5A), a Trichoderma reesei endoglucanase V (CEL45A), and aTrichoderma reesei endoglucanase III (CEL12A).
 4. The method of claim 1,which further comprises a GH61 polypeptide having cellulolytic enhancingactivity.
 5. The method of claim 1, wherein one or more of thecellulolytic enzymes, the CEL6 polypeptide having cellobiohydrolase IIactivity, and/or the GH10 polypeptide having xylanase activity are inthe form of a fermentation broth with or without cells.
 6. The method ofclaim 1, wherein the CEL6 polypeptide having cellobiohydrolase IIactivity has at least 96% sequence identity to the mature polypeptide ofSEQ ID NO:
 30. 7. The method of claim 6, wherein the CEL6 polypeptidehaving cellobiohydrolase II activity has at least 97% sequence identityto the mature polypeptide of SEQ ID NO:
 30. 8. The method of claim 7,wherein the CEL6 polypeptide having cellobiohydrolase II activity has atleast 98% sequence identity to the mature polypeptide of SEQ ID NO: 30.9. The method of claim 8, wherein the CEL6 polypeptide havingcellobiohydrolase II activity has at least 99% sequence identity to themature polypeptide of SEQ ID NO:
 30. 10. The method of claim 1, whereinthe CEL6 polypeptide having cellobiohydrolase II activity is encoded bya polynucleotide that hybridizes under very high stringency conditionswith the mature polypeptide coding sequence of SEQ ID NO: 29 or thefull-length complement thereof, wherein the very high stringencyconditions are defined as prehybridization and hybridization at 42° C.in 5×SSPE, 0.3% SDS, 200 μg/ml sheared and denatured salmon sperm DNA,and 50% formamide for 12 to 24 hours and washing three times each for 15minutes using 2×SSC, 0.2% SDS at 70° C.
 11. The method of claim 1,wherein the CEL6 polypeptide having cellobiohydrolase II activity isencoded by a polynucleotide having at least 96% sequence identity to themature polypeptide coding sequence of SEQ ID NO:
 29. 12. The method ofclaim 11, wherein the CEL6 polypeptide having cellobiohydrolase IIactivity is encoded by a polynucleotide having at least 97% sequenceidentity to the mature polypeptide coding sequence of SEQ ID NO:
 29. 13.The method of claim 12, wherein the CEL6 polypeptide havingcellobiohydrolase II activity is encoded by a polynucleotide having atleast 98% sequence identity to the mature polypeptide coding sequence ofSEQ ID NO:
 29. 14. The method of claim 13, wherein the CEL6 polypeptidehaving cellobiohydrolase II activity is encoded by a polynucleotidehaving at least 99% sequence identity to the mature polypeptide codingsequence of SEQ ID NO:
 29. 15. The method of claim 1, wherein the CEL6polypeptide having cellobiohydrolase II activity comprises the maturepolypeptide of SEQ ID NO:
 30. 16. The method of claim 1, wherein theCEL6 polypeptide having cellobiohydrolase II activity consists of themature polypeptide of SEQ ID NO:
 30. 17. The method of claim 1, whereinthe GH10 polypeptide having xylanase activity has at least 96% sequenceidentity to the mature polypeptide of SEQ ID NO:
 70. 18. method of claim17, wherein the GH10 polypeptide having xylanase activity has at least97% sequence identity to the mature polypeptide of SEQ ID NO:
 70. 19.The method of claim 18, wherein the GH10 polypeptide having xylanaseactivity has at least 98% sequence identity to the mature polypeptide ofSEQ ID NO:
 70. 20. The method of claim 19, wherein the GH10 polypeptidehaving xylanase activity has at least 99% sequence identity to themature polypeptide of SEQ ID NO:
 70. 21. The method of claim 1, whereinthe GH10 polypeptide having xylanase activity is encoded by apolynucleotide that hybridizes under very high stringency conditionswith the mature polypeptide coding sequence of SEQ ID NO: 69 or thefull-length complement thereof, wherein the very high stringencyconditions are defined as prehybridization and hybridization at 42° C.in 5×SSPE, 0.3% SDS, 200 μg/ml sheared and denatured salmon sperm DNA,and 50% formamide for 12 to 24 hours and washing three times each for 15minutes using 2×SSC, 0.2% SDS at 70° C.
 22. The method of claim 1,wherein the GH10 polypeptide having xylanase activity is encoded by apolynucleotide having at least 95% sequence identity to the maturepolypeptide coding sequence of SEQ ID NO:
 69. 23. The method of claim22, wherein the GH10 polypeptide having xylanase activity is encoded bya polynucleotide having at least 96% sequence identity to the maturepolypeptide coding sequence of SEQ ID NO:
 69. 24. The method of claim23, wherein the GH10 polypeptide having xylanase activity is encoded bya polynucleotide having at least 97% sequence identity to the maturepolypeptide coding sequence of SEQ ID NO:
 69. 25. The method of claim24, wherein the GH10 polypeptide having xylanase activity is encoded bya polynucleotide having at least 98% sequence identity to the maturepolypeptide coding sequence of SEQ ID NO:
 69. 26. The method of claim25, wherein the GH10 polypeptide having xylanase activity is encoded bya polynucleotide having at least 99% sequence identity to the maturepolypeptide coding sequence of SEQ ID NO:
 69. 27. The method of claim 1,wherein the GH10 polypeptide having xylanase activity comprises themature polypeptide of SEQ ID NO:
 70. 28. The method of claim 1, whereinthe GH10 polypeptide having xylanase activity consists of the maturepolypeptide of SEQ ID NO:
 70. 29. The method of claim 1, furthercomprising recovering the degraded cellulosic material.
 30. The methodof claim 29, wherein the degraded cellulosic material is a sugar. 31.The method of claim 30, wherein the sugar is selected from the groupconsisting of glucose, xylose, mannose, galactose, and arabinose.